JP2019510466A - Improved Enzymatic Modification of Wheat Phospholipids in Bread Application - Google Patents

Abstract

The present invention relates to a method for producing a dough comprising the steps of mixing a dough component, a phospholipase A2 enzyme acting on N-acylphosphatidylethanolamine at the sn2 position, and an enzyme acting on a polar lipid at the sn1 position. About. Also taught is a food grade enzyme composition comprising a phospholipase A2 enzyme capable of acting on N-acylphosphatidylethanolamine at the sn2 position and an enzyme acting on a polar lipid at the sn1 position.
[Selected figure] Figure 1

Description

CROSS-REFERENCES TO RELATED APPLICATIONS This application is US Provisional Patent Application No. 62 / 319,399, filed April 7, 2016, and December 2015, which are all incorporated herein by reference in their entirety. It is an international PCT application claiming the benefit of GB Patent 1522681.4 filed on the same day.

The present invention relates to novel enzyme combinations and their use in the manufacture of dough or baked products. The invention further relates to a method of producing a dough or baked product using a combination of novel enzymes.

  The lipids make up approximately 2% of the flour and these lipids are considered to be very important for the baking properties of the flour. Wheat flour lipids can be divided into non-polar and polar lipids, and it has been shown that the improved baking and baking properties are mainly due to the polar lipids.

  Over the past few decades, the increasing demand for consistent and higher quality baked goods has led to the use of a wide range of additives. It is known in the baking industry to supplement endogenous lipids, including naturally occurring polar lipids such as lecithin or additional enzymes such as, for example, lipases.

  One of the most abundant phospholipids in flour is N-acylphosphatidylethanolamine (NAPE), which is converted to N-acyl lysophosphatidylethanolamine (NALPE) by enzymatic hydrolysis. Further hydrolysis of NALPE produces N-acylglycerophosphoethanolamine (NAGPE).

  Accordingly, a first aspect of the present invention is a food grade enzyme composition comprising a phospholipase A2 enzyme acting on N-acylphosphatidylethanolamine at the sn2 position and an enzyme acting on a polar lipid at the sn1 position Providing a composition.

  In a further aspect, a method of producing a dough comprising mixing the dough component, a phospholipase A2 enzyme acting on N-acylphosphatidylethanolamine at the sn2 position, and an enzyme acting on a polar lipid at the sn1 position Is provided.

  In a still further aspect, the invention is the use of a phospholipase A2 enzyme acting on N-acylphosphatidylethanolamine at the sn2 position and an enzyme acting on a polar lipid at the sn1 position in the preparation of a dough or baked product, which is baked Product specific volume; to improve dough properties (dough formation; dough extensibility etc.); to improve the crispiness of the baked product crust; to improve the crumb structure (to improve the crumb diameter of the baked product) Or to improve the homogeneity of the crumb holes of the baked product); to improve the softness (such as to improve the softness of the baked product); to improve the oven spring of the baked product; Increase N-acyl lysophosphatidyl ethanolamine in baked products Increases N-acyl lysophosphatidylethanolamine having a fatty acid moiety containing 14 to 20 carbon atoms, preferably N-acyl lysophosphatidylethanolamine having a saturated fatty acid moiety containing 14 to 20 carbon atoms To increase the lysophospholipids in the dough and / or baked products; to increase the digalactosyl monoglycerides and / or monogalactosyl monoglycerides in the dough and / or baked products; in the dough and / or baked products And / or galactosyl monoglycerides and / or monogalactosyl monoglycerides together with the use thereof to increase N-acyl lysophosphatidylethanolamine.

  In yet a further aspect, a kit is provided comprising a set of phospholipase A2 enzymes capable of acting on N-acylphosphatidylethanolamine at the sn2 position, enzymes acting on polar lipids at the sn1 position, and instructions for use Ru.

  The invention still further provides a dough obtainable (preferably obtained) by the method according to the invention, or a baked product (preferably obtained) obtainable by the method according to the invention.

BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES The following sequences are described in Section 37 of the U.S. Regulatory Code 1.821-1.825 ("Requirements for Patent Applications Containing a Disclosure of Nucleotide and / or Amino Acid Sequences-Sequence In accordance with the World Intellectual Property Organization (WIPO) Standard ST. 25 (2009), the European Patent Convention and the Patent Cooperation Treaty (PCT), Rules 5.2 and 49.5 (a-bis), and the Sequence Listing requirements of Chapter 208 of the By-Laws and Appendix C. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 CFR 1.822.

  SEQ ID NO: 1 is the amino acid sequence of the enzyme in POWERBAKE® 4080 and POWERBAKE® 4090 acting on polar lipids at the sn1 position (US Pat. No. 8,012,732, incorporated herein by reference) Identical to SEQ ID NO: 6 from the specification). This enzyme is known to have both galactolipase and phospholipase activities.

  SEQ ID NO: 2 is the amino acid sequence of a mature lipid acyltransferase (GCAT) from Aeromonas salmonicida (see US Pat. No. 9,175,271).

  SEQ ID NO: 3 is the amino acid sequence of a phospholipase A2 enzyme that acts on N-acylphosphatidylethanolamine at the sn2 position found in MAXAPAL® A2.

  SEQ ID NO: 4 is the amino acid sequence of a phospholipase A2 enzyme (CRC08335) that acts on NAPE (N-acylphosphatidylethanolamine) at the sn2 position.

  SEQ ID NO: 5 is the nucleotide sequence of a phospholipase A2 enzyme (CRC08335) that acts on NAPE (N-acylphosphatidylethanolamine) at the sn2 position.

  SEQ ID NO: 6 is the N-terminal predicted signal peptide sequence of CRC08335.

Pomeranz, Y., in Modern Cereal Science and Technology ((1987) VCH Publishers, New York, NY). A list of polar and non-polar lipids found in flour (especially wheat flour) from Phospholipase A2 enzyme (SEQ ID NO: 3) that acts on N-acylphosphatidylethanolamine at the sn2 position when baking breads (MAXAPAL®), and an enzyme that acts on N-acylphosphatidylethanolamine at the sn1 position Figure 19 shows the softness effect observed when POWERBAKE (R) 4090) is used in combination. Phospholipase A2 enzyme (MAXAPAL®) that acts on N-acylphosphatidylethanolamine at the sn2 position when baking 100% whole wheat bread (MAXAPAL®), and an enzyme that acts on polar lipids at the sn1 position (POWERBAKE®) Fig. 4090) shows the softness effect observed when used in combination. Figure 19 shows the amino acid sequence (SEQ ID NO: 1) of POWERBAKE (R) 4080 and POWERBAKE (R) 4090 (both commercially available from DuPont Nutrition Biosciences ApS). SEQ ID NO: 4 shows the amino acid sequence of CRC08335. Figure 19 shows the nucleotide sequence of CRC08335 (SEQ ID NO: 5). FIG. 7 shows a plasmid map of pZKY512-1 containing the synthetic gene of CRC08335.

Abbreviation NAPE-N-Acylphosphatidylethanolamine NALPE-N-Acyllysophosphatidylethanolamine NAGPE-N-Acylglycerophosphoethanolamine DGDG-Digalactosyldiglyceride DGMG-Digalactosyl monoglyceride MGDG-Monogalactosyl diglyceride MGMG-Monogalactosyl monoglyceride PC-Phosphatidylcholine PLA-phospholipase A

  The inventive discovery of the present invention relates to a phospholipase A2 enzyme whose advantageous properties in foodstuffs (e.g. dough and / or baked products) act on N-acylphosphatidylethanolamine (NAPE) at the sn2 position, sn1 position Can be achieved by using in combination with enzymes that act on polar lipids.

  For the first time, we provide the synergy provided by the combination of a phospholipase A2 enzyme capable of acting on NAPE at the sn2 position, for example in foodstuffs such as dough or baked products, and an enzyme acting on polar lipids Indicated.

  Based on these discoveries, methods are provided for using phospholipase A2 enzymes capable of acting on NAPE at the sn2 position and enzymes acting on polar lipids at the sn1 position in the preparation of dough or products obtainable from the dough. The invention still further provides a food grade enzyme composition comprising a phospholipase A2 enzyme capable of acting on NAPE at the sn2 position and an enzyme acting on polar lipids at the sn1 position.

  The present invention relates to the dissolution of specific polar lipids in a special way in dough and food products obtainable from dough.

  Polar lipids contained in the majority of flours include phospholipids and galactolipids.

  A significant amount of phospholipid contained in flour, in particular wheat flour, may be N-acyl phosphatidyl ethanolamine (NAPE). Schafferczyk et al (J. of Agricultural and Food Chemistry (2014) 62: 8229-8237) show that flour contains on average 0.1% NAPE compared to 0.02% phosphatidylcholine (PC) Is teaching.

  Flour, in particular wheat flour, may contain galactolipids. For example, galactolipids such as digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride (MGDG) are naturally occurring (or endogenous) lipid constituents contained in flour, in particular wheat flour.

  Preferably, the phospholipid and / or galactolipid on which the enzyme used in the present invention acts is a naturally occurring phospholipid and / or galactolipid in powder.

  The phospholipase A2 enzyme that acts on NAPE at the sn2 position according to the present invention is an enzyme having PLA2 activity as taught herein "Assay for determining phospholipase activity and regiospecificity on NAPE".

Assays to Determine Phospholipase Activity and Regiospecificity on NAPE:
Substrate: 0.6% of 16: 0 to 18: 1 NAPE (N-Linoleoyl- (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) (requested to be obtained from Avanti, Or J. L. Newman et al., Chemistry and Physics of Lipids (1986) 42: 240-260), 0.4% TRITONTM-X 100 (Sigma Aldrich, St. Louis, MO) ; X-100) and deoxycholate in 0.003M also added to pancreatic enzymes and CaCl ₂ is dissolved in HEPES in buffer (pH 7.0) of 0.05M of 5 mM..

Assay procedure:
Incubate 2 mL substrate at 30 ° C., 0.1 mL enzyme solution in 0.05 M HEPES buffer (approximately 5 TIPU / mL or amount of enzyme corresponding to 2-5% substrate consumed 10 minutes after reaction) Was added and incubated at 30 ° C. for 10 minutes with magnetic stirring. Add 40 μL of 4 M HCl to stop the reaction and protonate free fatty acids. Add 1 mL of 99% ethanol and mix on a vortex mixer. 5 mL of MTBE (methyl tert-butyl ether) containing 0.5 mg of C17: 0 fatty acid (margaric acid) was added. The samples were again mixed on a vortex mixer for 5 seconds and extracted on a Rotamix at 25 rpm for 30 minutes. The sample was centrifuged at 1520 g for 10 minutes.

1 group of 500mg amine _(NH 2) -Bond Elut SPE column (Agilent Technologies, Santa Clara, CA ) placed on Bond Elut vacuum system. The column is conditioned using 8 mL of petroleum ether. Apply MTBE phase from the extraction onto the column,
First Fraction 8 mL of solvent A: MTBE: 2-propanol, 2: 1
Second fraction 8 mL of solvent B: acetone: formic acid 100: 2
Extract using

  The solvent was extracted at approximately 0.25 mL / min.

  The collected fatty acid fraction (second fraction) is evaporated to dryness and the fatty acid content is analyzed by GLC.

  Based on the internal standard C17: 0 fatty acids, the amount of C16: 0 and C18: 1 fatty acids is determined.

式中、
Ａ＝Ｃ１６：０脂肪酸（％）＋Ｃ１８：１脂肪酸（％）
２＝基質量（ｍＬ）
１００００００＝μｍｏｌへのモル変換
Ｄ＝酵素希釈係数
ＭＶ＝生成されたＣ１６：０脂肪酸およびＣ１８：１脂肪酸の平均分子量
１０＝反応時間（分間）
０．１＝アッセイに添加された酵素量（ｍＬ）
である。 Enzyme activity to NAPE is calculated as the amount of fatty acid (μmol) produced per minute under assay conditions.

During the ceremony
A = C16: 0 fatty acid (%) + C18: 1 fatty acid (%)
2 = base mass (mL)
1000000 = mol conversion to μmol D = enzyme dilution factor MV = average molecular weight of C16: 0 fatty acid and C18: 1 fatty acid formed 10 = reaction time (minutes)
0.1 = amount of enzyme added to the assay (mL)
It is.

Enzyme specificity is calculated as follows.

  For example, phospholipase A2 enzymes that preferentially dissolve, eg, hydrolyze, NAPE at the sn2 position are not only referred to in the present specification as “an assay for determining phospholipase activity and regiospecificity on NAPE”, at least to NAPE. It may be an enzyme with a relative PLA2 activity of more than 50%. An enzyme with a relative PLA2 activity of more than 50% means that this enzyme has a sn1 activity of less than 25% and a sn2 activity of more than 75%. Preferably, the phospholipase A2 enzyme which preferentially dissolves, e.g. hydrolyzes, NAPE at the sn2 position is relative as taught herein in the "Assay for determining phospholipase activity and regiospecificity on NAPE". It may be an enzyme having a relative PLA2 activity of at least 10% relative to PLA1 activity.

  Preferably, to determine that phospholipase A2 enzyme preferentially dissolves, eg, hydrolyzes NAPE at the sn2 position, as taught herein “for determining phospholipase activity and regiospecificity on NAPE Assay "is used. However, in some embodiments, this refers to whether the EnzChek phospholipase A2 assay kit from Invitrogen Product No. E10217 optionally reduces NAPE while increasing the formation of NALPE in the dough. It can be determined by using it together with the dough test to be analyzed.

  The term "especially" in connection with the phospholipase A2 enzyme that acts on NAPE at the sn2 position catalyzes only one specific reaction of NAPE at the sn2 position, such as dissolution (or hydrolysis), for this enzyme to generate 1-NALPE It means to do. A phospholipase A2 enzyme which specifically dissolves, eg hydrolyzes, NAPE at the sn2 position is compared to the PLA1 activity taught herein in the “Assay for determining phospholipase activity and regiospecificity on NAPE”. It may be an enzyme with a relative PLA2 activity of at least 80%. In one embodiment, the phospholipase A2 enzyme that acts on NAPE at the sn2 position according to the invention has the following enzymatic activity: phospholipase A2 activity (eg E.C. 3.1.1.4) or lipid acyltransferase activity (B) For example, it has one or more of E.C. 2.2.3.43).

  According to another embodiment, the phospholipase A2 enzyme that acts on NAPE at the sn2 position according to the invention is one that is capable of converting NAPE to 1-NALPE under dough conditions.

  According to another embodiment, the phospholipase A2 enzyme acting on NAPE at the sn2 position according to the invention converts NAPE to 1-NALPE, and the fatty acid part of the generated NALPE has 14 to 20 carbon atoms Contains

  In one embodiment, the phospholipase A2 enzyme that acts on NAPE at the sn2 position according to the invention is one that converts NAPE to 1-NALPE, and the fatty acid part of the generated NALPE is saturated and 14 to Contains 20 carbon atoms.

  In a further embodiment, the phospholipase A2 enzyme that acts on NAPE at the sn2 position according to the invention is that which converts NAPE to 1-NALPE, and the fatty acid moiety of the generated NALPE is saturated and 16 Contains a carbon atom (C16: 0). A phospholipase A2 enzyme that converts NAPE to 1-NALPE, wherein the fatty acid portion of the generated NALPE is saturated and contains 16 carbon atoms, is as taught herein “phospholipase activity and on NAPE Using an assay to determine regiospecificity and / or using the "HPLC / MS method for analyzing phospholipids extracted from dough" taught herein Can.

  In one embodiment, the use of the combination of enzymes according to the invention results in an amount of C16: 0 NALPE in the dough increased by at least 1.5 times compared to the dough without added enzyme. For example, the amount of C16: 0 NALPE in dough may be increased by at least 2.0 times, preferably at least 3.0 times.

  In one embodiment, the use of the combination of enzymes according to the invention increases the amount of C16: 0 NALPE in the dough increased by at least 1.5 to about 4.0 times compared to the dough without added enzyme Make it happen.

  Doe conditions are well known to those skilled in the art. These include the conditions during mixing of dough constituents or during standing and storage of dough. Suitable dough conditions include dough soaking, dough settling, dough scaling and dough shaping, and dough fermentation.

  According to another aspect of the present invention, the phospholipase A2 enzyme that acts on NAPE at the sn2 position claimed herein is unable or substantially unable to act on N-acyl lysophosphatidylethanolamine (NALPE) Can not act on

  As used herein, the term "cannot substantially act on N-acyl lysophosphatidylethanolamine" refers to "an assay for determining phospholipase activity and regiospecificity on NAPE" and "N-acyl lyso. Enzymes tested at the same dose both in the Assay to Determine Lysophospholipase Activity for Phosphatidylethanolamine (NALPE) mean that it has less than 20% activity for NALPE as compared to activity for NAPE Do. More preferably, enzymes which are substantially incapable of acting on N-acyl lysophosphatidylethanolamine have less than 10% activity on NALPE, more preferably 5% on NALPE, compared to activity on NAPE It has less activity, even more preferably less than 1% NALPE activity. Determination of the degree of saturation and length of fatty acids can be carried out by methods known in the art. Non-limiting examples are gas chromatography (GC) or liquid chromatography-mass spectrometry (HPLC / MS) as taught herein.

Assay to determine lysophospholipase activity towards N-acyl lysophosphatidylethanolamine (NALPE):
Substrate: 0.6% of 18: 1 NALPE (N-Linoleoyl- (1-oleyl-glycero-3-phosphoethanolamine) (requested from Avanti, or “N-acyl lysophosphatidyl ethanolamine ), 0.4% TRITONTM-X 100 (Sigma, X-100) and 5 mM CaCl ₂ dissolved in 0.05 M HEPES buffer (pH 7.0)). 0.003 M deoxycholate was also added to the pancreatic enzyme.

Assay procedure:
Incubate 2 mL substrate at 30 ° C., 0.1 mL enzyme solution in 0.05 M HEPES buffer (approximately 5 TIPU / mL or amount of enzyme corresponding to 2-5% substrate consumed 10 minutes after reaction) Was added and incubated with magnetic stirring for 10 minutes. 40 μL of 4 M HCl was added to quench the reaction and protonate free fatty acids. 1 mL of 99% ethanol was added and mixed with a vortex mixer. 5 mL of MTBE (methyl tert-butyl ether) containing 0.5 mg of C17: 0 fatty acid (margaric acid) was added. The samples were again mixed on a vortex mixer for 5 seconds and extracted on a Rotamix at 25 rpm for 30 minutes. The sample was centrifuged at 1520 g for 10 minutes.

One 500 mg amine (NH2) -Bond Elut SPE column (Agilent) was placed on a Bond Elut vacuum system. The column was conditioned with 8 mL of petroleum ether. Apply MTBE phase from the extraction onto the column,
First Fraction 8 mL of solvent A: MTBE: 2-propanol, 2: 1
Second fraction 8 mL of solvent B: acetone: formic acid 100: 2
Extracted using

  The solvent was extracted at approximately 0.25 mL / min.

  The collected fatty acid fraction (fraction 2) was evaporated to dryness and the fatty acid content was analyzed by GLC.

  Based on the internal standard C17: 0 fatty acid, the amount of C18: 1 is determined.

式中、
Ａ＝Ｃ１８：１脂肪酸（％）
２＝基質量（ｍＬ）
１００００００＝μｍｏｌへのモル変換
Ｄ＝酵素希釈係数
ＭＶ＝Ｃ１８：１脂肪酸の分子量
１０＝反応時間（分間）
０．１＝アッセイに添加された酵素量（ｍＬ）
である。 Enzyme activity to NALPE is calculated as fatty acid (μmol) produced per minute under assay conditions.

During the ceremony
A = C18: 1 fatty acid (%)
2 = base mass (mL)
1000000 = molar conversion to μmol D = enzyme dilution factor MV = molecular weight of C 18: 1 fatty acid 10 = reaction time (minutes)
0.1 = amount of enzyme added to the assay (mL)
It is.

Synthesis of N-acyl lysophosphatidylethanolamine (NALPE) 1.5 g of 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18: 1 Lyso PE from Avanti) in 50 mL chloroform Dissolve and add 550 μL of triethanolamine and coat under nitrogen. The solution was cooled on an ice bath and 1.9 g of linoleic anhydride was added dropwise while stirring. The solution was coated under nitrogen and allowed to react at 22 ° C. for 20 hours. The crude reaction product was concentrated by evaporating chloroform under vacuum and purified by column chromatography. The reaction product N-linoleoyl-1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (NALPE) was isolated and the structure confirmed by NMR and HPLC / MS.

  In one embodiment, the phospholipase A2 enzyme that acts on NAPE at the sn2 position according to the invention may be MAXAPAL®, LYSOMAX® Oil, Pancreatic PLA2, Lipomod 699L from Biocatalysts.

  Enzymes that act on polar lipids at the sn1 position according to the invention can be assayed as follows: "Assays for determining enzymes that act on polar lipids (phospholipases) at the sn1 position" and / or "polar lipids (galactolipids at the sn1 position" (galactolipids) An enzyme that acts on polar lipids at the sn1 position, determined using one or both of the following “assays to determine the enzyme that acts on MGDG)”.

Assay to determine enzymes that act on polar lipids (phospholipases) at the sn1 position:
Phospholipase A1 activity (PLA1) is a substrate of PED-A1 (N-((6- (2,4-DNP) amino) hexanoyl) -1- (BODIPY® FL C5) -2-hexyl-Sn- as substrate. It was measured using glycero-3-phosphoethanolamine (A10070, manufactured by ThermoFisher Scientific).

  This substrate is specific for PLA1 and is dye-labeled glycerophosphoethanolamine with BODIPY® FL dye-labeled acyl chain and dinitrophenyl quencher modified head group at the sn1 position. The quenching efficiency is reduced by cleavage of the BODIPY® FL pentanoic acid substituent at the sn1 position and with the enzyme resistant ether bond at the sn2 position.

  The result is a PLA1 dependent increase in fluorescence emission detected at 515 nm.

procedure:
The “lipid mix” was prepared by mixing 30 μL of 10 mM dioleoylphosphatidyl choline in ethanol, 30 μL of 10 mM dioleoylphosphatidyl glycerol in ethanol, and 30 μL of 2 mM PED-A1 in DMSO.

  Add 5 mL of buffer (50 mM Tris HCl), 0.14 mM NaCl and 2 mM CaCl2 (pH 7.4) to a 20 mL beaker. Stir using a magnetic stirrer to form a vortex. To form a matrix liposome mix, slowly add 50 μL of the lipid mix to the side of the vortex over 1 minute using a 100 μL pipette equipped with a narrow gel loading tip with an opening.

  Add 50 μL of enzyme sample (or standard or control) and 50 μL of substrate liposome mix to microtiter plate wells. Protect from light and incubate at room temperature for 30 minutes. The fluorescence is measured using a microtiter plate reader, with excitation at 470 nm and emission at 515 nm.

  Calibration curves are constructed based on multiple standard PLA1 solutions of various enzyme concentrations from 0 to 10 U / mL. The enzyme standard is PLA1 (L3295 from Sigma) with known activity. Based on the fluorescence measurement of the standard solution, a calibration curve of fluorescence intensity as a function of enzyme concentration U / mL was constructed. The activity of the unknown sample was measured (U / mL) based on the standard curve.

Assay for determining the enzyme acting on polar lipids (galactolipids; MGDG) at the sn1 position Substrate: 0.6% 1-linoleyl-2-oleyl-3-O-(-D-galactopyranosyl)- Dissolve sn glycerol (C18: 2, C18: 1 MGDG), 0.4% TRITONTM-X 100 (Sigma, X-100) and 5 mM CaCl ₂ in 0.05 M HEPES buffer (pH 7) I did. 0.003 M deoxycholate was also added to the pancreatic enzyme.

Assay procedure:
Incubate 2 mL of substrate at 30 ° C., 0.1 mL of enzyme solution in 0.05 M HEPES buffer (approximately 2 to 5 TIPU / mL or enzyme corresponding to approximately 5% substrate consumed by reaction for 10 minutes) Was added and incubated at 30 ° C. for 10 minutes with magnetic stirring. 40 μL of 4 M HCl was added to quench the reaction and protonate free fatty acids. 1 mL of 99% ethanol was added and mixed with a vortex mixer. 5 mL of MTBE (methyl tert-butyl ether) containing 0.5 mg of C17: 0 fatty acid was added. The samples were again mixed for 5 seconds on a Wortex mixer and extracted over 30 minutes on a Rotamix at 25 rpm. The sample was centrifuged at 1520 g for 10 minutes.

One 500 mg amine (NH2) -Bond Elut SPE column (Agilent) was placed on a Bond Elut vacuum system. The column is conditioned using 8 mL of petroleum ether. The MTBE phase from the extraction is applied on the column and
First Fraction 8 mL of solvent A: MTBE: 2-propanol, 2: 1
Second fraction 8 mL of solvent B: acetone: formic acid 100: 2
Extracted using

  The solvent was extracted at approximately 0.25 mL / min.

  The collected fatty acid fraction (fraction 2) was evaporated to dryness and fatty acids were analyzed by GLC. The amounts of C18: 2 and C18: 1 fatty acids were determined based on the internal standard C17: 0 fatty acids.

式中、
Ａ＝Ｃ１８：２脂肪酸（％）＋Ｃ１８：１脂肪酸（％）
２＝基質量（ｍＬ）
１００００００＝μｍｏｌへのモル変換
Ｄ＝酵素希釈係数
ＭＶ＝生成されたＣ１８：２脂肪酸およびＣ１８：１脂肪酸の平均分子量
１０＝反応時間（分間）
０．１＝アッセイに添加された酵素量（ｍＬ）
である。 Enzyme activity was calculated as fatty acid (μmol) produced per minute under assay conditions.

During the ceremony
A = C18: 2 fatty acid (%) + C18: 1 fatty acid (%)
2 = base mass (mL)
1000000 = mol conversion to μmol D = enzyme dilution factor MV = average molecular weight of C18: 2 fatty acids and C18: 1 fatty acids formed 10 = reaction time (minutes)
0.1 = amount of enzyme added to the assay (mL)
It is.

Enzyme specificity is calculated as follows.

Synthesis of 1-Linoleyl-2-oleyl-3-O-(-D-galactopyranosyl) -sn glycerol (C18: 2, C18: 1 MGDG).
1-Linoleyl-2-oleyl-3-O-(-D-galactopyranosyl) -sn glycerol (C18: 2, C18: 1 MGDG)
1-monolinoleyl-2-hydroxy-3-O-(-D-2 ', 3', 4 ', 6'-tetra-O-acetylgalactopyranosyl) -sn-glycerol is described in Selmair and Koehler (J. Agric. Food Chem. (2008) 56: 6691-6700). 1-monolinoleyl-2-hydroxy-3-O-(_- D-2 ', 3', 4 ', 6'-tetra-O-acetylgalactopyranosyl) -sn-glycerol is obtained by column chromatography 99 Isolated and purified to greater than% purity.

  Acylation of the sn2 position of 1-monolinoleyl-2-hydroxy-3-O-(-D-2 ', 3', 4 ', 6'-tetra-O-acetylgalactopyranosyl) -sn-glycerol is It was carried out according to the method of Gaffney and Reese (J. Chem. Soc., Perkin Trans. (2001) 1: 192-205) using oleic acid as the acyl donor.

  Deacylation of 1-linoleoyl-2-oleyl-3-O-(-D-2 ′, 3 ′, 4 ′, 6′-tetra-O-acetylgalactopyranosyl) -sn-glycerol was carried out in methanol And then purified by column chromatography, and the structure is confirmed by mass spectroscopy and NMR analysis.

  In one embodiment, an enzyme that acts on polar lipids at the sn1 position according to the present invention is more than a relative sn2 activity in an assay entitled “An assay for determining an enzyme that acts on polar lipids (phospholipase) at the sn1 position”. It is an enzyme with at least 20% higher relative sn1 position activity. In one embodiment, an enzyme that acts on polar lipids at the sn1 position according to the present invention is more than a relative sn2 activity in an assay entitled “An assay for determining an enzyme that acts on polar lipids (phospholipase) at the sn1 position”. It is an enzyme with at least 50% higher relative sn1 activity.

  In one embodiment, the enzyme that acts on polar lipids at the sn1 position according to the invention is more than the relative sn2 activity in an assay entitled “Assay for Determining an Enzyme That Acts on Polar Lipids (MGDG) at the sn1 Position”. It is an enzyme with at least 20% higher relative sn1 position activity. In one embodiment, the enzyme that acts on polar lipids at the sn1 position according to the invention is more than the relative sn2 activity in an assay entitled “Assay for Determining an Enzyme That Acts on Polar Lipids (MGDG) at the sn1 Position”. It is an enzyme with at least 50% higher relative sn1 activity.

  In one embodiment, the enzyme that acts on polar lipids at the sn1 position according to the invention is an enzyme capable of hydrolyzing at least 10% of DGDG using HPTLC analysis of dough lipids in dough.

  In one embodiment of the present invention, the enzyme acting on polar lipids at the sn1 position is an enzyme having phospholipase activity, galactolipase activity or a combination thereof.

  The term "polar lipid" means a polar lipid found in flour, preferably flour. Polar lipids found in wheat flour are described by Pomeranz, Y. et al. Defined above (above; see FIG. 1). In one embodiment, the term "polar lipid" means one or more of the group consisting of phospholipids, galactolipids or combinations thereof. The phospholipid may be one or more of phosphatidyl choline, N-acyl phosphatidyl ethanolamine, phosphatidyl ethanolamine, phosphatidyl serine or phosphatidyl inositol. In one embodiment, preferably, the phospholipid may be phosphatidyl choline. The galactolipid may be one or more of digalactosyl diglyceride, ceramide diglyceride, 6-o-acetyl steryl glucoside or ceramide diglucoside. In one embodiment, preferably, the galactolipid may be diphosphatidyl diglyceride. In one embodiment, the enzyme that acts on polar lipids at the sn1 position is phospholipase A1, eg having phospholipase A1 activity, E. coli. C. It can be classified as 3.1.1.32.

  An enzyme that acts on polar lipids at the sn1 position can act on galactolipids such as digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride (MGDG). This may be added to its phospholipase A1 activity.

  Thus, in one embodiment, the enzyme acting on polar lipids at the sn1 position is, for example, galactolipase and E. coli. C. It can be classified as 3.1.1.26.

  In a further embodiment, the enzyme that acts on polar lipids at the sn1 position acts on DGDG at the sn1 position.

  As used herein, the term "acting on" in relation to enzymes that act on polar lipids at the sn1 position, means that the enzyme removes fatty acids from the sn1 position of the polar lipids (eg by hydrolysis), and thus For example, it means releasing free fatty acids.

  The term "preferentially" in relation to an enzyme that acts on polar lipids at the sn1 position refers to the hydrolysis of polar lipids at the sn1 position as compared to, for example, catalyzing the dissolution of polar lipids at the sn2 position. It means to give priority to catalyzing decomposition. Enzymes that act on polar lipids at the sn1 position are assays: "Assays to determine enzymes that act on polar lipids (phospholipase) at the sn1 position", and / or "phospholipase activity and sn1 and sn2 to PC (phosphatidylcholine) And / or an assay for determining an enzyme that acts on a polar lipid (galactolipid; MGDG) at the sn1 position.

Assay for Determining Phospholipase Activity and Regiospecificity of sn1 and sn2 to PC (Phosphatidylcholine) Substrate: 0.6% 16: 0 to 18: 1 PC, 1-palmitoyl-2-oleoyl-sn-glycero -3-Phosphocholine (Avanti Polar Lipids Inc., Alabama, Alabama; Product No. 850457), 0.4% TRITONTM-X 100 (Sigma, X-100) and 5 mM CaCl ₂ in 0.05 M HEPES It was dissolved in buffer (pH 7). 0.003 M deoxycholate was also added to the pancreatic enzyme.

Assay procedure:
Incubate 2 mL of substrate at 30 ° C., 0.1 mL of enzyme solution (approximately 2-5 TIPU / mL in 0.05 M HEPES buffer or enzyme corresponding to 2-5% substrate consumed after 10 minutes of reaction) Amount) was added and incubated with magnetic stirring for 10 minutes. Add 40 μL of 4 M HCl to stop the reaction and protonate free fatty acids. Add 1 mL of 99% ethanol and mix on a vortex mixer. 5 mL of MTBE (methyl tert-butyl ether) containing 0.5 mg of C17: 0 fatty acid (margaric acid) was added. The samples were again mixed for 5 seconds on a vortex mixer and extracted over 30 minutes on a Rotamix at 25 rpm. The sample was centrifuged at 1520 g for 10 minutes.

1 group of 500mg of amine _(NH 2) -Bond Elut SPE column (Agilent) was placed on a Bond Elut vacuum system. The column is conditioned using 8 mL of petroleum ether. The MTBE phase from the extraction is applied on the column and
First Fraction 8 mL of solvent A: MTBE: 2-propanol, 2: 1
Second fraction 8 mL of solvent B: acetone: formic acid 100: 2
Extracted using

  The solvent was extracted at approximately 0.25 mL / min.

  The collected fatty acid fraction (fraction 2) was evaporated to dryness and fatty acids were analyzed by GLC. Based on the internal standard C17: 0 fatty acids, the amount of C16: 0 and C18: 1 fatty acids is determined.

式中、
Ａ＝Ｃ１６：０脂肪酸（％）＋Ｃ１８：１脂肪酸（％）
２＝基質量（ｍＬ）
１００００００＝μｍｏｌへのモル変換
Ｄ＝酵素希釈係数
ＭＶ＝生成されたＣ１６：０脂肪酸およびＣ１８：１脂肪酸の平均分子量
１０＝反応時間（分間）
０．１＝アッセイに添加された酵素量（ｍＬ）
である。 Enzyme activity is calculated as fatty acid (μmol) produced per minute under assay conditions.

During the ceremony
A = C16: 0 fatty acid (%) + C18: 1 fatty acid (%)
2 = base mass (mL)
1000000 = mol conversion to μmol D = enzyme dilution factor MV = average molecular weight of C16: 0 fatty acid and C18: 1 fatty acid formed 10 = reaction time (minutes)
0.1 = amount of enzyme added to the assay (mL)
It is.

Enzyme specificity is calculated as follows.

  The term "preferentially" in relation to an enzyme that acts on polar lipids at the sn1 position refers to the hydrolysis of polar lipids at the sn1 position as compared to, for example, catalyzing the dissolution of polar lipids at the sn2 position. It means to give priority to catalyzing decomposition. Enzymes that act on polar lipids at the sn1 position are assays: "Assays to determine enzymes that act on polar lipids (phospholipase) at the sn1 position", and / or "phospholipase activity and sn1 and sn2 to PC (phosphatidylcholine) And / or an assay for determining an enzyme that acts on a polar lipid (galactolipid; MGDG) at the sn1 position.

  An enzyme that preferentially acts on polar lipids at the sn1 position is “an assay for determining phospholipase activity and sn1 and sn2 positional specificity for PC (phosphatidylcholine)” and / or “polar lipid at the sn1 position (galactolipids) Means that the relative PLA1 / sn1 activity will be at least 60%, as determined using the “assay to determine the enzyme acting on MGDG)”.

  In one embodiment, an enzyme that preferentially acts on polar lipids at the sn1 position is “an assay for determining phospholipase activity and sn1 and sn2 position specificity for PC (phosphatidylcholine)”, and / or “sn1 position It means that relative PLA1 / sn1 activity will be at least 70%, as determined using “an assay for determining enzymes that act on polar lipids (galactolipids; MGDG)”.

  The term "specifically" in the context of an enzyme that acts on polar lipids at the sn1 position means that this enzyme will only catalyze the hydrolysis of polar lipids at the sn1 position.

  An enzyme that specifically acts on polar lipids at the sn1 position is “an assay for determining phospholipase activity and sn1 and sn2 positional specificity for PC (phosphatidylcholine)” and / or “polar lipid at the sn1 position (galactolipids) Means having at least 60% (preferably at least 70%) relative PLA1 / sn1 activity, as determined using “an assay for determining an enzyme acting on MGDG)”.

  According to one embodiment, the enzyme that acts on polar lipids at the sn1 position can comprise the enzyme taught in WO 02/03805 (incorporated herein by reference). In one embodiment, enzymes that act on polar lipids at the sn1 position include: POWERBAKE® 4080, POWERBAKE® 4090, PANAMORE®, LIPOPAN FTM and LIPOPAN EXTRATM included.

  In one embodiment, the enzyme that acts on polar lipids at the sn1 position may be a phospholipase A1 from Fusarium oxysporum (e.g. LIPOPAN F (TM)). In one embodiment, phospholipase A1 from Fusarium oxysporum may be the enzyme taught in WO 98/26057, which is incorporated herein by reference.

  In one embodiment, the enzyme acting on polar lipids at the sn1 position according to the invention has at least 60% sequence identity with SEQ ID NO: 1, more preferably at least 70%, at least 80%, at least 90%, at least 95% Or an enzyme with 100% identity.

  Preferably, the enzyme that acts on polar lipids at the sn1 position has low activity for NAPE.

  One advantage of the present invention is the use of a combination of a phospholipase A2 enzyme that acts on N-acylphosphatidylethanolamine (NAPE) at the sn2 position and an enzyme that acts on polar lipids at the sn1 position.

  The fatty acid compositions of phospholipids and galactolipids at the sn1 and sn2 positions differ significantly in both fatty acid length and saturation level.

  Surprisingly, by combining enzymes acting at either the sn1 or sn2 position, for example to the dough and the products obtained from the dough by cooking such as baking, steaming, boiling or frying. It is possible to obtain combinations of polar lipids such as, for example, lithophospholipids and lithogalactolipids, such as MGMG or DGMG, which provide significant advantages.

  Even in NAPE, the fatty acids present at the sn1 or sn2 position are generally quite different, with more unsaturated fatty acids being found at the sn2 position. Phospholipase A1 can hydrolyze NAPE at the sn1 position to generate 2-NALPE using, for example, a fatty acid at the sn2 position (Structural Analysis of Wheat Flour Glycolipids. Lipids, Vol. 6, No. 10 , P. 768-776)).

  For this reason, the present invention has the effect of dissolving (eg, hydrolyzing) NAPE at the sn2 position in combination with modifying a polar lipid (eg, an additional polar lipid) with an enzyme acting at the sn1 position. About.

  We show, for the first time, the importance of selectively dissolving (eg, hydrolyzing) NAPE and additional polar lipids.

  According to the invention, the phospholipase A2 enzyme and the enzyme acting on polar lipids are at least 10 of the baked product compared to the baked product manufactured under the same conditions except the addition of the enzymes claimed herein. The dough components are mixed in an effective amount to produce an increase in specific volume which is%.

  According to the invention, the phospholipase A2 enzyme acting on N-acylphosphatidylethanolamine at the sn2 position and the enzyme acting on the polar lipid at the sn1 position exclude the addition of the enzymes claimed herein of the baked goods Dough composition in an effective amount to produce an increased softness which is at least 5%, preferably at least 10%, more preferably at least 20%, most preferably at least 30% compared to a baked product produced under the same conditions Mixed into ingredients.

  The terms "improved softness" and "increased softness" as used herein are considered synonymous and may mean a decrease in force per specific volume in a baked product.

  Preferably, the food-grade enzyme composition comprises a lipid component from a dough or baked product (e.g. gas chromatography (GC) or liquid chromatography-mass spectroscopy (HPLC / MS) or HPTLC analysis) When extracted, an increase of more than about 0.005% w / w (of monogalactosyl monoglyceride based on dry dough weight), preferably compared to the same dough or baked product when no enzyme is added An increase of more than 0.01% w / w (of monogalactosyl monoglyceride based on dry dough weight), preferably an increase of more than 0.025% w / w, preferably an increase of 0.05% w / w , Preferably showing an increase of more than 0.075% w / w (of monogalactosyl monoglyceride based on dry dough weight), dough or baked It is believed to increase the monogalactosylated monoglyceride content in the goods.

  Preferably, the food-grade enzyme composition comprises a lipid component from a dough or baked product (e.g. gas chromatography (GC) or liquid chromatography-mass spectroscopy (HPLC / MS) or HPTLC analysis) When extracted, greater than about 0.005 to 0.1% w / w (based on dry dough weight) of monogalactosyl monoglyceride as compared to the same dough or baked product when no enzyme is added It is believed to show an increase and to increase the monogalactosyl monoglyceride content in the dough or baked product.

  According to the present invention, the food grade enzyme composition has lipid components from dough or baked products (e.g. gas chromatography (GC), or liquid chromatography-mass spectroscopy (HPLC / MS), or HPTLC analysis) 2.) at least 5%, preferably at least 10%, more preferably at least 20% of the content of digalactosyl diglycerides, as extracted, compared to the same dough or baked product when the food grade enzyme composition is not mixed More preferably, it exhibits a reduction of at least 30%, preferably at least 40%, and is believed to reduce the digalactosyl diglyceride content in the dough or baked product.

  According to the invention, the food grade enzyme composition is believed to reduce digalactosyl diglycerides in the dough or baked products. When the lipid component is extracted from a fully proofed dough or baked product (eg, subjected to gas chromatography (GC) or liquid chromatography-mass spectroscopy (HPLC / MS) or HPTLC analysis), it is for food use. As compared to the same dough or baked product when the enzyme composition was not mixed, at a relative digalactosyl diglyceride content (eg, about 0.01 to 0.1% w / w based on dry dough) It is analyzed to show a reduction of 5% to 50%.

  In one embodiment, the phospholipase A2 enzyme of the invention is present at a concentration of 100 to 7500 ePLU / kg powder. In one embodiment, the phospholipase A2 enzyme is administered at 150-2000 ePLU / kg powder.

  In one embodiment, the enzyme that acts on polar lipids at the sn1 position of the present invention is present at a concentration of 50-2000 TIPU / kg (powder). In one embodiment, the enzyme that acts on polar lipids at the sn1 position of the present invention is administered at a concentration of 200 to 800 TIPU / kg (powder).

ePLU assay:
Phospholipase A2 enzyme (ePLU) can be determined using the following assay using egg yolk as a substrate.

  This assay is performed according to the Food Chemicals Standard Chemicals (FCC, 8 ed., Appendix 5 p. 1328).

Substrate:
44 g of egg yolk (2 eggs yolk in one beaker) was added to 200 mL of water and homogenized using an Ultra Turrax mixer. 10 mL of 0.3 M calcium chloride was added. Ten mL of substrate was transferred to a titration glass and 10 mL water and 5 mL of 0.016 M deoxycholate were added. The substrate was incubated at 40 ° C. and the pH was adjusted to pH 8 with 0.05 M NaOH using a pH stat titrator. 0.1 M enzyme was added and titration data collected over 5 minutes. The titrant was 0.05 M NaOH. Activity (ePLU) was calculated as fatty acid released per minute (μmol) under assay conditions, using the slope for titrant consumption (70 seconds to 170 seconds) as a function of time.

TIPU assay:
Phospholipase activity (TIPU) can be determined using the following assay:
Substrate: 0.6% L-alpha phosphatidyl choline 95% plant (Avanti, Product No. 441601), 0.4% TRITONTM-X 100 (Sigma, X-100) and 5 mM CaCl ₂ 0.05 M Dissolved in HEPES buffer (pH 7).

Assay procedure:
Samples, calibration and control samples were diluted in 10 mM HEPES (pH 7.0) containing 0.1% TRITONTM X-100. The analysis was performed using a Konelab Autoanalyzer (Thermo, Finland). The assay was run at 30 ° C. 34 μL of substrate was thermostated at 30 ° C. for 180 seconds, after which 4 μL of enzyme sample was added. Enzymization lasted for 600 seconds. The amount of free fatty acid liberated during the enzymization was measured using a NEFA kit obtained from Wako Chemicals GmbH, Germany).

This assay kit consists of two reagents.
NDFA-HR (1):
0.53 U / mL of acyl-CoA synthase (ACS)
0.31 mM coenzyme A (CoA)
4.3 mM adenosine 5-triphosphate disodium salt (ATP)
1.5 mM 4-amino-antipyrine (4-AA)
2.6 U / mL ascorbic acid oxidase (AOD)
50 mM phosphate buffer (pH 7.0) containing 0.062% sodium azide
NDFA-HR (2):
2.4 mM 3-Methyl-N-ethyl-N- (E-hydroxyethyl) -aniline (MEHA)
12 U / mL of acyl-CoA oxidase (ACOD)
14 U / mL peroxidase (POD)

  After enzymeification, 113 μL of NEFA-HR (1) was added and the mixture was incubated for 300 seconds. Thereafter, 56 μL of NEFA-HR (2) was added and the mixture was incubated for 300 seconds. Next, the OD520 nm was measured. Enzyme activity (μmol (FFA) / min · mL) was calculated based on a calibration curve generated from oleic acid. The enzyme activity TIPU (pH 7) was calculated as fatty acid (μmol) produced per minute under the assay conditions.

  The flour dough may not contain all of the sufficient amount of lipid substrate for the composition of the present invention. For this reason, it is within the scope of the invention to supplement the dough with at least one galactolipid, phospholipid or combination thereof to provide a sufficient substrate for the enzyme. The expression "sufficient substrate" will be understood to mean that none of the lipid substrates is limiting in order to obtain the dough improving action or the baked product improving action described above.

  Auxiliary lipid substrates for the enzymes of the invention may be polar lipids. Particularly useful lipids in this connection are oils or fats of cereal origin, such as, for example, oat oil. Oat oil typically contains 5-25% phospholipids and 5-12% glycolipids in addition to triglycerides. Oat oil can be fractionated to produce fractions with high content of polar lipids.

  Thus, it is contemplated that one or more phospholipids can be added to the dough. In this regard, useful phospholipids include phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), phosphatidyl glycerol (PG) and phosphatidyl choline (PC).

  In one embodiment, the composition of the present invention further comprises lecithin.

  In a further embodiment, the composition for use according to the present invention further comprises lecithin.

  In another embodiment, the method of the present invention further comprises the step of mixing lecithin.

  In one embodiment, the lecithin of the invention is soy bean derived lecithin.

  In another embodiment, the lecithins of the invention are enzymatically modified.

  Preferably, the lecithins of the invention are enzymatically modified by an enzyme with phospholipase A2 activity.

  Preferably, the lecithins of the invention are modified by phospholipase A2 which can act at the sn2 position of N-acetylphosphatidylethanolamine.

  The present invention is the use of a phospholipase A2 enzyme acting on N-acylphosphatidylethanolamine at the sn2 position and an enzyme acting on a polar lipid at the sn1 position in the preparation of a dough or baked product, the specific volume of the baked product; To improve the properties of the dough (dough formation; dough extensibility etc); to improve the crispiness of the baked product crust; to improve the crumb structure (to improve the crumb diameter of the baked product or to make the baked product To improve the homogeneity of the crumb holes of the body); to improve the softness (to improve the softness of the baked product); to improve the oven spring of the baked product; N in the dough and / or the baked product -Increase acyl lysophosphatidyl ethanolamine (preferably 14 to 20 charcoals Increasing N-acyl lysophosphatidyl ethanolamine with fatty acid moieties containing atoms, preferably increasing N-acyl lysophosphatidyl ethanolamine with saturated fatty acid moieties containing 14 to 20 carbon atoms; And / or to increase the lysophospholipids in the baked products; to increase the digalactosyl monoglycerides and / or monogalactosyl monoglycerides in the dough and / or baked products; lysophospholipids in the dough and / or baked products, and / or Further provided is the use for increasing N-acyl lysophosphatidylethanolamine together with increasing digalactosyl monoglycerides and / or monogalactosyl monoglycerides.

  The present invention further advantageously provides improved quality features (improved specific volume of baked products, improved crusty crispiness, etc .; improved crumb structure (improved crumb diameter of baked products or baked products) Crumb hole homogeneity etc.); improved softness (such as improved softness of baked products); improved capping of baked products; improved oven springs of baked products) Do.

  Thus, in one embodiment, the method of the invention comprises the further step of baking the dough to obtain a baked product. One particularly desirable property of baked bread products is the high specific volume specified in the examples. Thus, the addition of the enzyme of the invention preferably results in an increase of the specific volume of the baked product which is at least 10% compared to the baked product produced under the same conditions except that no enzyme is added. More preferably, the specific volume increase is at least 20%, such as at least 30%, such as at least 40%.

  It is known in the art that enzymes other than lipase can contribute to improved dough properties and quality of baked products. It is within the scope of the present invention that in addition to the composition of the invention at least one further enzyme can be added. Such further enzymes include lipases, starch degrading enzymes (eg amylase or amyloglucosidase), hemicellulases (eg xylanases), cellulases, oxidoreductases (eg glucose oxidases such as hexose oxidase), lipid acyltransferases, Includes branch enzymes (eg, pullulanase), lactase and proteases.

  According to another embodiment, the method claimed herein comprises an additional step in which additional enzymes are mixed into the dough component.

Specific Volume The specific volume in a baked product can be defined as the volume of the product divided by its weight (g / mL or g / ccm).

  The present invention relates to improving the specific volume of baked products.

Dough Characteristics The present invention may relate to dough characteristics such as dough formation; improving dough extensibility. The present invention does not adversely affect the stickiness of the dough.

  These can be measured in the dough as follows.

Crustiness of crusts The present invention may be related to improving the crispiness of crusts of baked products.

  This can be measured in baked products, such as bread or rolls, as described below.

Crumb structure The present invention may relate to improving the crumb structure, such as improving the crumb diameter of the baked product or improving the homogeneity of the crumb pores of the baked product.

  These can be measured in baked products, such as bread or rolls, as described below.

Softness The present invention may relate to improving softness (such as improving the softness of baked products).

  Softness can be measured by any method known in the art.

  This can be measured in baked products, such as bread or rolls, as described below.

  In one embodiment, the softness (or hardness) of the sliced bread was determined from texture profile analysis (TPA) using a texture analyzer TAXT Plus from Stable Microsystems. As an example, a 35 mm metal probe can be used to measure softness on day 1 (D1) and day 3 (D3).

Capping The present invention does not adversely affect the capping of baked products. One general firing feature known as "capping" is commonly found and is particularly undesirable. Capping occurs when the top solidifies (i.e. hardens) and then this top is pushed up causing the batter to leak out of the interior of the baked goods, e.g. muffins or rolls. The result is an undesirable baked product such as muffins or rolls.

  Capping was assessed subjectively by testing the baked products, and the amount of capping observed was assigned a qualitative number.

  This can be measured in a baked product as follows.

Oven Spring The present invention may relate to improving the oven spring of a baked product. As used herein, the term "oven spring" means a rapid increase (rise) in volume of baked goods, such as bread, when they are placed in a hot oven.

  This can be measured in a baked product as follows.

Increasing or improving As used herein, the term "increasing or improving" refers to a dough obtainable from said dough (e.g. a baked product) but without the addition of an enzyme according to the invention Or to increase or improve as compared to the product.

  An additional technical function of the present invention is to increase N-acyl lysophosphatidyl ethanolamine in dough and / or baked products (preferably N-acyl lyso with fatty acid moieties containing 14 to 20 carbon atoms) Increasing phosphatidyl ethanolamine, preferably N-acyl lysophosphatidyl ethanolamine with a saturated fatty acid moiety containing 14 to 20 carbon atoms); lyso-phospholipids in dough and / or baked products Increasing; including increasing digalactosyl monoglycerides and / or monogalactosyl monoglycerides in the dough and / or baked products.

  In one preferred embodiment, the invention relates to N-acyl lysophosphatidyl ethanol along with increasing lyso-phospholipids and / or digalactosyl monoglycerides and / or monogalactosyl monoglycerides in a dough and / or baked product It relates to increasing amines (NALPE) (preferably 1-NALPE).

Synergism / synergy The inventors for the first time, for example, with phospholipase A2 enzymes capable of acting on NAPE at the sn2 position in foodstuffs such as dough or baked products, and enzymes acting on polar lipids at the sn1 position. The synergy provided by the combination of

  The terms "synergism" or "synergism" mean an increase in action (eg, volume of bread) greater than the increase obtained from each enzyme when used individually or separately at the same dose.

Food or Groceries The methods, uses or compositions of the invention can be used in the preparation of foodstuffs. Here, the term "foodstuff" is used in a broad sense and includes foodstuffs for humans and foodstuffs for animals (i.e. feedstuffs). In one preferred aspect, the foodstuff is for human consumption.

  In the present invention, the term "dough component" refers to any composition comprising flour (eg flour, preferably flour), any one of water or yeast, or one or more of flour, water and / or yeast. Means

  Preferably, the enzyme of the present invention is mixed with dough components.

  Preferably, the enzyme of the present invention is mixed with flour or a composition comprising flour.

  In one embodiment, the comestible product is a dough or a product made from dough, for example by cooking or cooking such as baking or steaming, boiling or frying.

  In one embodiment, a baked product is obtainable (preferably obtained) from dough.

  In one embodiment, the steamed product is available from dough (preferably obtained).

  In one embodiment, the boiled product is available from dough (preferably obtained).

  In one embodiment, the fried product is available from dough (preferably obtained).

  In one embodiment, the food product is a baked product.

  In one embodiment, the foodstuff is a steamed product.

  In one embodiment, the food product is a chew product.

  In one embodiment, the foodstuff is a fried product.

  The method, use or composition of the present invention may be used in the preparation of a dough or a product made from the dough, for example by cooking, for example baking, steaming, boiling or frying heating.

  Preferably, a baked product is produced by baking the dough produced according to the invention.

  Preferably, the product is produced by kneading the dough produced according to the invention.

  Preferably, the steamed product is produced by steaming the dough produced according to the invention.

  Preferably, the fried product is produced by frying the dough produced according to the present invention.

  For certain embodiments, preferably the foodstuff is a baked good, such as bread (eg white bread, whole grain bread or rye bread; typically a loaf of bread or rolls, French baguette style bread, Pita bread, flatbread, crisp bread or pizza bread), tortilla, pancake, muffin, pie crust, pastry, Danish pastry, cake, biscuit or cookie.

  In one aspect, the foodstuff is a steamed product, such as steamed bread, dumplings.

  In one aspect, the foodstuff is a boiled product, such as noodles or pasta.

  In one aspect, the comestible is a fried product, such as a donut.

  The "food grade enzyme composition" as defined herein may be any composition suitable for being added to the dough or suitable for mixing with the dough components.

  As one non-limiting example, the food products of the present invention include baked products and dough products.

  As used herein, the term "dough" refers to a highly viscous, malleable mixture of flour and liquid (eg, water). The dough can contain yeast or other leavening agents. The dough can further comprise other dough components such as, for example, fats, or flavorings, or salts, or sugars.

  The dough according to the invention can be produced from one or more of flours selected from wheat flour, corn flour, rice flour, rye flour, leguminous flour, almond flour or other flours.

  In one embodiment, the dough is manufactured from wheat flour.

  The methods and uses of the present invention may be part of any baking process. The composition of the present invention can be used in any baking process. As an example, the baking process may be one or more processes selected from the group consisting of sponge and dough; straight; no-time and continuous bread making.

"Sponge and dough" method Without being limited by theory, the sponge and dough grinding method may be composed of two separate steps, a sponge step and a dough step. In the first stage (sponge stage), the sponge is manufactured and fermented for a period of time, and in the second stage (dough stage), the sponge is added to the final dough component which results in the full formulation. Other terms for "sponge" include yeast starter or yeast pre-fermented species. In French-style bread, the sponge and dough method may be known as levain-levure.

  In the first step, a mixture called sponge is used to make about 1/3 to 3/4 flour, yeast, yeast food (eg sugar) and malt (malt), and hard dough or more liquid brewe It may contain sufficient water. Although shortening may be added at this stage, shortening is usually added later and 1/3 to 3/4 of salt may be added to control the fermentation.

  The sponges may be mixed in any suitable spin-up apparatus, preferably equipped with a temperature control device.

  Suitably, this may be a large horizontal dough mixer processing 1 ton per batch, optionally constructed with a heat exchange jacket to allow temperature control.

  The goal of the mixing process is the "formation" of the dough into an elongated, intertwined protein network that will form a near homogeneous blend of ingredients by the formation of gluten and the basic structure of a loaf of bread. Since extreme shear action has to be avoided, a conventional dough mixer will rotate slowly at 35-75 revolutions per minute, oriented parallel to the body of the mixer, and their action will cause the dough to Has several parallel bars extending and kneading. A typical dip cycle will be about 12 minutes.

  The mixed sponge is dumped into a bowl, which is a shallow rectangular metal tank on the wheel, and placed in an area of controlled temperature and humidity (eg, 27 ° C. and 75% relative humidity) where Fermented until the volume starts to decrease. The time required for this process, called drop or break, is frequently adjusted to produce a drop in about 3 to 5 hours, eg temperature, type of flour, amount of yeast, absorption and malt Depends on variables such as the amount of

  In the second or dough stage, the sponge is returned to the mixer and the remaining ingredients are added. The dough is stretched to an optimal consistency and then returned to the fermentation chamber or placed at "floor time" for more maturation.

  In one embodiment, the phospholipase A2 enzyme acting on the N-acylphosphatidylethanolamine at the sn2 position and the enzyme acting on the polar lipid at the sn1 position are added either at the sponge stage or at the dough stage, preferably at the sponge stage Good. These may be added simultaneously or sequentially.

  In another embodiment, a phospholipase A2 enzyme that acts on N-acylphosphatidylethanolamine at the sn2 position may be added to the sponge stage (e.g., during mixing flour and other dough components). Alternatively or additionally, an enzyme that acts on polar lipids at the sn1 position may be added to the dough stage (e.g. during the kneading step).

  In one embodiment, a phospholipase A2 enzyme capable of acting on NAPE is added to the sponge and an enzyme acting on polar lipids at the sn1 position is added to the dough.

  In one embodiment, lecithin, preferably a soy based lecithin, may be added in addition to the sponge stage (e.g. during the process of mixing flour and other dough components). Preferably, lecithin may be added together with at least one phospholipase A2 enzyme that acts on N-acylphosphatidylethanolamine at the sn2 position. Suitably, the lecithin may be an enzymatically modified lecithin. In one embodiment, lecithin may be enzymatically modified by an enzyme with phospholipase A2 activity (preferably lecithin is enzymatically modified by phospholipase A2 acting on N-acylphosphatidylethanolamine at the sn2 position Good).

  In one embodiment, phospholipase A2 or a portion thereof is added during the sponge step.

The "straight dough" method The straight dough method may be a simple mixing process of baking. All ingredients (eg, ingredients) for making the dough are all placed together and combined in a single kneading or tanning session. After raising, bulk fermented rest is done before splitting.

  In one embodiment, a phospholipase A2 enzyme that acts on N-acylphosphatidylethanolamine at the sn2 position and an enzyme that acts on a polar lipid at the sn1 position may be mixed with the dough component. These may be added simultaneously or sequentially.

  In one embodiment, lecithin, preferably soy based lecithin may be added additionally.

  Suitably, the lecithin may be enzymatically modified lecithin. In one embodiment, lecithin may be enzymatically modified by an enzyme with phospholipase A2 activity (preferably lecithin is enzymatically modified by phospholipase A2 acting on N-acylphosphatidylethanolamine at the sn2 position Good).

"No-time" method The "no-time" method is a special subset of the straight dough method. By way of non-limiting example, increased amounts of yeast, and fast-acting oxidizing agents such as, for example, ascorbic acid and azodicarbonamide, allow elimination of most of the bulk fermentation period of straight dough.

Continuous Baking Although the numerous steps in the preparation and configuration of conventional dough have been fully automated, none of the processes are truly continuous. In a continuous system, the dough is handled without interruption from the time the ingredients are mixed until it is deposited in the pan. The initial fermentation process is still essentially a batchwise procedure, but in continuous baking lines the conventional sponge is replaced with a liquid pre-fermented species called broth or brew. The brew is composed of a mixture of water, yeast, sugar and flour and some of the other ingredients, fermented for several hours before being mixed into the dough.

  After the brew has finished fermentation, the brew, along with the dry ingredients, is fed to a spinner which mixes all the ingredients into a homogenous mass. A batter-like material passes through a dough pump that regulates the flow rate and delivers the mixture to the deployment apparatus where the kneading operation is applied. The expansion tank is an important device in a continuous line.

  About 50 kg (100 lbs) of processing can be done for 90 seconds each, which has a batter with no tissue structure, little extensibility, and smooth out of fluid clumps with inadequate gas residue Change to dough to form an elastic film. The dough is then transferred from the spreading tank into the weighing device which constantly extrudes the dough, intermittently cuts it into loaf-sized chunks and drops it into a container passing underneath.

  In one embodiment, the phospholipase A2 enzyme acting on the N-acylphosphatidylethanolamine at the sn2 position, and the enzyme acting on the polar lipid at the sn1 position are for example post-fermentation and during baking of the dough into liquid pre-fermented species or dough May be added to These may be added simultaneously or sequentially.

  In another embodiment, a phospholipase A2 enzyme that acts on N-acylphosphatidylethanolamine at the sn2 position may be added to the liquid pre-fermented species. Alternatively or additionally, an enzyme that acts on polar lipids at the sn1 position may be added to the dough after fermentation and during kneading of the dough.

  In one embodiment, lecithin, preferably a soy based lecithin, may be additionally added to the pre-fermentation stage or to the dough, for example either after fermentation of the dough and during grinding. Preferably, lecithin may be added together with at least one phospholipase A2 enzyme that acts on N-acylphosphatidylethanolamine at the sn2 position. Suitably, the lecithin may be enzymatically modified lecithin. In one embodiment, lecithin may be enzymatically modified by an enzyme with phospholipase A2 activity (preferably lecithin is enzymatically modified by phospholipase A2 acting on N-acylphosphatidylethanolamine at the sn2 position Good).

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al. , DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED. John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those skilled in the art with many general dictionaries of terms used in this disclosure.

  The present disclosure is not limited by the exemplary methods and materials disclosed herein, but any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments of the present disclosure. It can be used. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise stated, all nucleic acid sequences are written left to right 5 'to 3'; amino acid sequences are written left to right amino to carboxy, respectively.

  The headings provided herein are not limited to the various aspects or embodiments of the invention that can be obtained by reference to the specification as a whole. Thus, the terms defined immediately below are more particularly defined by reference to the entire specification.

  Amino acids are referred to herein using the three letter abbreviation or the single letter abbreviation amino acid nomenclature.

  The term "protein" as used herein includes proteins, polypeptides and peptides.

  The term "amino acid sequence" as used herein is synonymous with the term "polypeptide" and / or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme".

  The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional one and three letter codes for amino acid residues can be used. The three letter code for amino acids is as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide can be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

  Other definitions of terms may appear throughout the specification. Before describing the exemplary embodiments in more detail, it is to be understood that the present disclosure is not limited to the particular embodiments described, which can, of course, vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting as the scope of the present disclosure is limited only by the appended claims. Should.

  Where a range of numerical values is provided, each intervening value between the upper and lower limit values of the range is also specified to the first decimal place of the unit of the lower limit, unless the context clearly indicates otherwise. Are understood to be disclosed in Each narrower range between any indicated values or intervening values within the indicated range and any other indicated or intervening values within the indicated range are encompassed by the present disclosure. The upper and lower limits of these narrower ranges may independently be included or excluded within that range, either or both limits are included within the narrower ranges, or both limits are Each range not included is also included in the present disclosure on the premise that any limit is specifically excluded in the display range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present disclosure.

  As used in this specification and the appended claims, the singular forms “one (a)”, “an” and “the” indicate that the context of the claim is otherwise It should be noted that the plural referents are also included unless otherwise specified. Thus, for example, reference to "enzyme" includes a plurality of such candidate substances, and reference to "dough components" refers to one or more dough components known to those skilled in the art and the like, and the like. Reference to the equivalent of

  The publications discussed herein are provided solely in the sense of being disclosed prior to the filing date of the present application. In this specification, such publications should not be construed as an admission that they constitute prior art to the claims appended hereto.

  The invention will now be described, by way of example only, with reference to the following examples.

Materials and methods:
material:
-A phospholipase A2 enzyme which is commercially available from DSM, NL and which acts on N-acylphosphatidylethanolamine, in particular at the sn2 position-MAXAPAL®, # 4313 (10000 ePLU / mL)
LIPOMODTM 699L is a phospholipase A2 enzyme that acts on NAPE at the sn2 position, which is a pancreatic phospholipase A2 commercially available from Biocatalysts (12000 ePLU / mL).
· POWERBAKE ® 4080 (commercially available from DuPont), an enzyme that acts on polar lipids at the sn1 position-This enzyme is known to have both galactolipase activity and phospholipase activity (10000 TIPU / g ), The amino acid sequence shown in SEQ ID NO: 1.
· POWERBAKE ® 4090 (commercially available from DuPont), an enzyme that acts on polar lipids at the sn1 position-This enzyme is known to have both galactolipase activity and phospholipase activity (15,500 TIPU / G), which has the amino acid sequence shown in SEQ ID NO: 1 (POWERBAKE 4090 is the same enzyme as POWERBAKE 4080, but POWERBAKE 4090 is more concentrated in enzyme ).
LYSOMAX® Oil, a glycerophospholipid cholesterol acyl transferase (GCAT) otherwise known as lipid acyltransferase (SEQ ID NO: 2; commercially available from Dupont). This enzyme can act on N-acylphosphatidylethanolamine at the sn2 position. Enzyme activity, 1000 TIPU / g.
LIPOPANTM is an enzyme that acts on polar lipids at the sn1 position and is called phospholipase A1. This enzyme is commercially available from Novozymes DK (18500 TIPU / g).
-PANAMORE (TM) Golden Conc. , # 4240 is an enzyme that acts on polar lipids at the sn1 position, and is called phospholipase A1. This enzyme is commercially available from DSM, NL (29000 TIPU / g).
SUREBAKE® 800, hexose oxidase (HOX) from Dupont.

TLC analyzer:
Applicator: Automatic TLC sampler 4, CAMAG ADC2 automated development chamber programmed to an elution time of 7 cm.
HPTLC plate: 10 × 20 cm silica plate No. 1 from Merck. 1.05641.0001. Activated on a CAMAG TLC plate heater III for 10 minutes at 160 ° C. before use.
Development: The HPTLC silica plate was dried on a CAMAG TLC plate heater III for 10 minutes at 160 ° C., cooled and immersed in 6% copper acetate in 16% H ₃ PO ₄ . It was further dried at 160 ° C. for 6 minutes and evaluated immediately thereafter.

Analysis of dough lipids:
A sample of 5 μL of dough lipid (from 200 mg of dried dough) dissolved in 0.5 mL of heptane: isopropanol (3: 2) was applied to the HPTLC plate.
Standard substance 1A: 0.1% DGDG (digalactosyl diacylglycerol) (Avanti, product no. # 840524) dissolved in heptane: isopropanol (3: 2) in various amounts (( Applied at 0.5-1-2-3 and 5 .mu.L).
Running buffer 4 to 1: chloroform: methanol: water (192: 78: 12) (standard running buffer for dough lipids).
Running buffer in water 6: methyl acetate: 1-propanol: methanol: 0.25% KCl (25: 25: 25: 10: 9) (used to separate more phospholipids).
10 mL of running buffer in the plate chamber and 25 mL in the filter paper chamber.
After development, the TLC chromatogram was scanned and areas of different components were calculated using a CAMAG TLC scanner.
A calibration curve was constructed based on the area of the DGDG standard and the individual components of the powdered lipid were calculated based on this calibration curve.

P-NMR analysis:
Lecithin sample (40 ± 5 mg) in 1 mL of 4: 2: 3 CDCl ₃ : MeOH: CsCDTA (aq) (deuterated chloroform: methanol: cesium-1,2-diaminocyclohexanetetraacetic acid, volume / volume) It was dissolved. The CDTA solution was prepared with 1 M concentration in Milli-Q water. Next, CsOH · H ₂ O (cesium hydroxide: water) was added to adjust the pH to 10.5. The sample was vortexed for 10 seconds, centrifuged at 4500 rpm for 10 minutes at 20 ° C., then 550 μL was transferred to a 5 mm NMR tube using a 1000 μL Hamilton syringe and placed in the NMR instrument for analysis.

  Triisobutyrate phosphate was used as an internal standard (2 mg). We found that the NMR spectrum collected at 5 ° C. produces an ideal spectrum with optimal peak width and signal dispersion.

  NMR spectra were obtained using a Bruker Advance III spectrometer (Faellanden, Switzerland), SampleJet sample changer (Bruker, Faellanden, Switzerland) and a 5 mm BBO (Broadband Observe) probe (Bruker, Faellanden, Switzerland) tuned to phosphorus. , Collected under automation at 14.1T. Spectra were collected under quantitative conditions.

LC / MS Analysis of Phospholipids Extracted from Dough Dough lipid samples were full scan m by using heated electrospray in positive and negative mode by liquid chromatography coupled with triple quadrupole mass spectrometer online / Z 50-1500 was analyzed. NALPE is deprotonated ion [M-H] in negative mode ^- was formed.

  The column was a normal phase column (DIOL) and the mobile phase was acetonitrile / acetone (80/20) to which 20 mL of water was added in 1 L. The water contained 5 mM ammonium formate.

  The sample was dissolved in 2 mL of acetonitrile / acetone (80:20). Trace amounts of selected NALPE were extracted and the areas compared.

Instrument Agilent 1100
Binary pump (G1312B) + μ-vacuum degassing agent (G1379B)
High-performance autosampler ALS (G1367E) + thermostat 1290 (G1330B)
Column section (G1316A)
A triple quadrupole mass spectrometer (MS7) manufactured by TSQ Vantage, Thermo Finnigan, equipped with a heated electrospray interface (HESI-II).
Column: YMC Pack Diol 120 S-5 μm, 12 nm 4.6 × 50 mm (# 526)

Chromatography conditions

Sample Preparation 0.2 g of dried dough-derived lipid was added to 2 mL of acetonitrile / acetone (80:20) and sonicated for 10 minutes.

  The mixture was centrifuged at 16,000 g for 3 minutes and the supernatant was injected as it is.

  Calculation: Based on HPLC / MS analysis, determine the amount of NALPE component with various fatty acids, C16: 0 NALPE, C18: 0 NALPE, C18: 1 NALPE, C18: 2 NALPE, C18: 3 The relative content of NALPE was calculated.

Extraction of dough lipid:
The fully proofed dough samples were immediately frozen in liquid nitrogen. The dough was then frozen and freeze dried. The dried dough was finely ground and sieved. 1.0 g of sample was weighed into a 15 mL centrifuge tube with a lid. 7.5 mL of water saturated butanol (WSB) was added and mixed on a vortex mixer. The sample was placed in a 90 ° C. water bath for 10 minutes and then placed on a RotaMix (25 rpm) for 30 minutes. The sample was again placed in a 90 ° C. water bath for 10 minutes and then placed on a RotaMix for 30 minutes. The samples were centrifuged at 2000 rcf for 10 minutes. 1.5 mL of the organic phase was removed to a small tumbler, evaporated to dryness under nitrogen vapor and used for any further analysis.

Example 1
Firing Experiment Testing POWERBAKE® 4080 in Combination with LYSOMAX® Oil In this experiment, POWERBAKE® 4080 was tested in combination with LYSOMAX® Oil in a recipe for hard crust roll.

  Kneading on a Diosna spiral mixer. Water uptake for flour according to analysis: 400 BU-2%.

Procedure Mix all ingredients at a slow speed for 1 minute in a bowl-add water and knead slowly for 2 minutes and at high speed for 6.5 minutes. The dough temperature should be around 26 ° C. Measure 1350 g of dough and mold it by hand. The dough is placed in a heating cabinet at 30 ° C. for 10 minutes.

  The dough is formed into 30 dough balls on a "Glimik (TM) rounder" in a setting according to the table on the machine.

  The dough was proofed at 34 ° C., 85% relative humidity for 45 minutes and baked for 5 minutes at 200 ° C./2 L steam for 13 minutes + damper open for 5 minutes (MIWE oven program 1). After firing, the rolls were cooled at ambient temperature for 25 minutes and then weighed to measure volume.

  The characteristics of dough and bread are evaluated by those skilled in the art.

  The experimental configuration and the results from the firing evaluation are shown in Table 1.

  The fully fermented dough was frozen and freeze dried, and the lipids in the dried dough were extracted with water saturated butanol and analyzed by HPTLC.

  The components from the HPTLC analysis were quantified based on the calibration curve for DGDG analyzed on the same plate using the results shown in Table 2.

  The bake test results in Table 1 illustrate that the addition of POWERBAKE 4080 to the dough has a strong effect on the bread volume. However, the addition of LYSOMAX® Oil in combination with 30 ppm Powerbake further increases the bread volume and a clear synergy is seen. This synergy is also considered as an improvement in dough and bread scores. 100 ppm LYSOMAX® Oil gives the most potent synergy, and HPTLC analysis shows that POWERBAKE® 4080 is a monogalactosyl diglyceride (while forming monogalactosyl monoglyceride (MGMG) and digalactosyl monoglyceride (DGMG) It is established that both MGDG) and digalactosyl diglyceride (DGDG) have potent activity. This enzyme also has potent activity at NAPE during the formation of NALPE. LYSOMAX® Oil also has activity on NAPE during formation of NALPE. Some activity towards galactolipids in dough of LYSOMAX® Oil is also seen.

  Without wishing to be bound by theory, the synergistic firing performance between POWERBAKE® 4080 and LYSOMAX® Oil shows that LYSOMAX® Oil is active on the sn2 position of NAPE. Although, on the other hand, POWERBAKE® is due to the fact that it is active at the sn1 position in polar lipids (including MGDG and DGDG and phospholipases including NAPE).

Example 2
Firing experiments testing LYSOMAX® Oil at doses of 25 ppm to 200 ppm Example 1 showed that the optimum dose of LYSOMAX® Oil was 100 ppm in combination with POWERBAKE® 4080. To test the dose response in detail, LYSOMAX® Oil was tested in combination with POWERBAKE® 4080 at doses of 25 ppm to 200 ppm. The results are shown in Table 3.

  The bake test results confirm the synergy of 30 ppm POWERBAKE 4080 in combination with LYSOMAX® Oil. At least 75 ppm of LYSOMAX® Oil is required to see synergy and optimal dose at 100 ppm.

  The fully fermented dough was frozen and freeze dried, and the lipids in the dried dough were extracted with water saturated butanol and analyzed by HPTLC (Table 4).

  Analysis of dough lipids confirms the activity of LYSOMAX® Oil to NAPE, but further decreases the dose of NALPE with increasing doses of LYSOMAX® Oil, and NAGPE is formed It is also clear. Doses of 25-200 ppm of LYSOMAX® Oil have only a very slight effect on galactolipids.

  The dough lipids were also analyzed by P-NMR focusing on the isomeric composition of NALPE shown in Table 5. The results from Table 5 show that as the dose of LYSOMAX® Oil increases, more 1-NALPE is generated due to the sn2 specificity of this enzyme.

Example 3
Baking experiment to test POWERBAKE® 4080 in combination with MAXAPAL® In this experiment, POWERBAKE® 4080 is combined with MAXAPAL® or LYSOMAX® Oil in hard crust roll recipe It tested together.

  MAXAPAL® is a phospholipase with high PLA2 specificity.

  The experimental configuration and the results from the firing evaluation are shown in Table 6.

The total dough and bread scores are calculated as the sum of the individual scores, separate from the sticky scores, added as (10-tackiness).
Experiment 1: Total score = 7 + (10-7) + 4 + (10-6) + 5 + 5 + 4 + 5 + 5 = 42

  The results from Table 6 clearly illustrate that the combination of POWERBAKE® 4080 and Maxapal produces a pronounced synergy with respect to bread volume, and that the dough and bread characteristics are also improved . In addition, it is also observed that MAXAPAL® is extremely resistant to various doses, where the increased effect is seen from the 100-750 ppm dose of MAXAPAL®.

  The fully fermented dough was frozen and freeze dried, and the lipids in the dried dough were extracted with water saturated butanol and analyzed by HPTLC.

  Components from HPTLC analysis were quantified based on the calibration curve for DGDG analyzed on the same plate using the results shown in Table 7.

  HPTLC analysis confirms that MAXAPAL® is extremely active in NAPE during formation of NALPE.

  This enzyme is very specific for NAPE and no significant formation of NAGPE is observed. This may explain why MAXAPAL is resistant to different doses. MAXAPAL® has no activity on DGDG, but exemplified its small activity on MGDG as MGDG formation was observed. Without wishing to be bound by theory, the specificity of MAXAPAL® for the hydrolysis of NAPE to NALPE is the reason why this enzyme has positive synergy in combination with POWERBAKE® 4080 In addition, it also explains why MAXAPAL® can not be easily overdosed. However, it is clear that the combination of POWERBAKE® 4080 and MAXAPAL® produces a small amount of NAGPE.

  This means that MAXAPAL® produces sn1-NALPE, which is a more preferred substrate for POWERBAKE® 4080 than sn2-NALPE, because POWERBAKE® 4080 is active on fatty acids at the sn1 position. It can be explained by the fact that

  In order to examine in more detail the specificity of these enzymes and the effect on fatty acid composition of NALPE in dough, lipids extracted from dough were analyzed by HPLC / MS, C16: 0, C18: 0, C18: The relative composition of NALPE with 1, C18: 2 and C18: 3 fatty acid compositions was analyzed. Based on the fatty acid composition of NALPE in the dough and the amount of NALPE calculated from TLC analysis of the dough lipid, the relative amount of C16: 0_NALPE in the dough was calculated as shown in the table shown in Table 8 (control dough The relative amount of C16: 0_NALPE in was defined as 100%).

  The results in Tables 7 and 8 confirm that POWERBAKE® 4080 produces significant amounts of NALPE in dough, but the amount of C16: 0_NALPE increases only slightly (3-8 %). This is explained by the fact that POWERBAKE® 4080 hydrolyzes fatty acids at the sn1 position of NAPE. When the dough is treated with MAXAPAL®, the amount of NALPE in the dough is also increased, in which case MAXAPAL® C16 to hydrolyze fatty acids at the sn2 position of NAPE in the dough. It is observed that the amount of: 0_NALPE increases strongly (340%).

  Combining POWERBAKE® 4080 with MAXAPAL® can increase the amount of C16: 0_NALPE, and as shown in Table 7, this enzyme combination is in the process of manufacturing DGMG and MGMG. It is also active on galactolipids such as DGDG and MGDG in dough.

  The positive synergy that POWERBAKE® 4080 and MAXAPAL® have on baking performance was explained by the combined action on galactolipids and NAPE during formation of DGMG, MGMG and 16: 0_NALPE. MAXAPAL® is additionally active against other phospholipids such as PC and PE in dough, and it is known that these components also have more saturated fatty acids at the sn1 position. For this reason, LPC and LPE produced in dough are also predicted to have higher amounts of saturated (c16: 0) fatty acids.

Example 4
Baking experiments with hard crustol The purpose of this experiment is to test LIPOMOD 699 L, another PLA2 from Biocatalysts, and to investigate its combined action with the sn1 specific enzyme POWERBAKE 4080 Was that.

  These enzymes were tested according to the procedure for hard crust roll (Example 1) to evaluate the specific volume of bread and the properties of dough and bread.

  These enzymes were tested as outlined in Table 9, but the bake test results are also shown in Table 9.

  The results from Table 9 clearly show the synergy of combining PLA2 LIPOMODTM 699 L with the sn1 specific glycolipase POWERBAKE 4080. LIPOMODTM 699 L by itself increases the bread volume slightly, and POWERBAKE® 4080 also significantly increases the bread volume. However, the combination of these two enzymes increases the volume of bread more compared to the individual enzymes. In addition, the total dough and bread scores are also significantly improved by combining these two enzymes.

Example 5
Firing experiment using hard crust roll.
Previous bake tests have shown that the PLA2 enzyme exhibits synergy when combined with the sn1 specific enzyme POWERBAKE® 4080. This enzyme also has sn1 specific phospholipase activity. In this test, other sn1 specific phospholipases were tested in combination with MAXAPAL®, PLA2 as shown in Table 10.

  These enzymes were tested according to the procedure for hard crustol (example 1), but modified with the new reform powder (DK2015-00040).

  The bake test results obtained by combining MAXAPAL (R) with PANAMORE (R) and LIPOPAN F (TM) clearly showed synergy to the bread volume.

Example 6
Firing experiment using hard crust roll.
Baking experiments show that the combination of the sn1-specific enzyme POWERBAKE® 4080 and the sn2-specific enzyme MAXAPAL® has a positive synergy in the volume of bread when used in baking. There is. However, it is known that the amount of phospholipid in the flour is rather limited. The purpose of this study was to investigate the action of these enzymes when soy lecithin was incorporated into the dough.

  The calcination experiment was carried out according to the procedure of hard crustol (Example 1) using enzymes and lecithin shown in Table 11.

  The effect on bread volume shown in Table 11 clearly confirms the synergy of POWERBAKE® 4080 and MAXAPAL® in dough containing 0.2% or 0.5% lecithin ing.

Example 7
Firing experiment using hard crust roll.
In this baking experiment, the action of MAXAPAL® PLA2 and glycolipase POWERBAKE® 4080 in hard crustol using powder made in the US called Polar Bear (DK2015-00071) was tested. Baking experiments were performed according to the procedure of hard crustol (Example 1) except that no fungal alpha amylase was added. The configuration and results of the experiment are shown in Table 12.

  The bake test results from Table 12 confirm the synergy of the combination of MAXAPAL® PLA2 and glycolipase POWERBAKE® when these enzymes were tested in US milling.

Example 8
Baking experiments with sponge and dough bread The baking experiments show that it is possible to obtain positive synergy of sn1 and sn2 with activity on NAPE in the dough. This positive effect may be explained by the production of NALPE with saturated fatty acid (C16: 0_NALPE) at the glycerol moiety.

  However, these two types of enzymes, when added to the dough, compete with the NAPE substrate, so the combination of the two enzymes uses sn2-specific phospholipase (MAXAPAL®) alone It has been observed to produce fewer C16: 0 NALPEs than would be the case.

  However, in certain baking procedures, such as the sponge and dough procedure, it is possible to add the enzyme on both the sponge side and the dough side. If the sn2-specific phospholipase is added to the sponge side only, no competition with NAPE substrate will occur. It is envisaged herein that sn1 specific glycolipase can be added on the dough side.

  Another aspect of adding the enzyme on the sponge side is that functional polar components formed during sponge fermentation can be utilized during dough grinding.

  L. Gerits et al. According to (Food Chemistry 172 (2015) 613-621), the addition of emulsifying agents like DATEM to the dough had an influence on the rheology of the dough, while on the other hand the addition of lipase fermented the hydrolyzed lipids There was no effect as it was released only to significant levels during.

  The addition of enzymes to the sponge in the sponge and dough making procedure forms a hydrolyzed lipid with positive functional rheological properties in the dough.

  In the following firing test, MAXAPAL (R) was added to the sponge and POWERBAKE (R) 4080 was added to the dough side. Baking experiments were performed according to the sponge and dough procedure taught below using reform powder (DK2015-00040).

sponge:
1) Mix all ingredients for 1 minute at first speed-Mix for 3 minutes at second speed in a Hobart mixer.
2) The temperature of the sponge should be around 25.5 ° C.
3) Ferment the sponge at 30 ° C., 85% RH for 3 hours-an open bowl.

Doe:
1) Mix all remaining ingredients except sponge and salt in a Hobart mixer for 2 minutes at low speed and 5 minutes at medium speed (use ice water).
2) Add salt-Mix at medium speed for 8 minutes.
3) Measure a 550g dough.
4) Let the dough stand for 10 minutes at ambient temperature.
5) Molding on a Glimek molder: 1: 4-2: 3-3: 15-4: 12-Width: 8 on both sides.
6) Place the formed dough in a tin can.
7) Proof for 60 minutes at 43 ° C., 95% RH.
8) Bake for 26 minutes at 200 ° C. (Miwe oven, program 4).
9) Remove the pan from the tin can, cool for 70 minutes, weigh and measure the volume.

  The dose of the enzyme and the results of the baking test are shown in Table 13.

  The results in Table 13 show that when MAXAPAL (R) is added to the sponge and POWERBAKE (R) is added to the dough, MAXAPAL (R) and POWERBAKE (R) 4080 positive in the sponge and doughpan procedure. It is confirmed that it is possible to obtain synergy of

Example 9
Firing Experiment with Sponge and Dough Pan The sponge and dough pan manufacturing procedure has been used conventionally and is still used extensively in the US bread industry. The sponge and dough procedure is characterized by a two step dough kneading process. The sponge is made by kneading flour (70% of total flour), water and yeast, which are fermented for a very long time (3 hours). The sponge is then mixed with the remaining flour, water, sugar, salt and other ingredients. Usually, when adding enzymes to the dough but adding two enzymes that will compete against the same substrate, add one enzyme to the sponge side and then the other enzyme to the dough side be able to.

  In the following experiments, MAXAPAL (R) and POWERBAKE (R) 4080 were tested using U.S. powder (Polar Bear # DK2015-00071) in a sponge and dough making procedure, and for these enzymes a table is shown. Test results as shown in 14 were obtained.

  The results from Table 14 confirm that MAXAPAL® added to the sponge, when combined with POWERBAKE® 4080 added to the dough, produces a synergy on the volume of the bread.

Example 10
Firing experiments with sponge and dough For the effect of the addition of MAXAPAL® in combination with POWERBAKE® 4080, sponge and dough using MAXAPAL® added to either the sponge side or the dough side The bread procedure was investigated in detail. The configuration and results of the experiment are shown in Table 15.

  The bread volume results presented in Table 15 confirm that positive synergy was obtained by adding the combination of MAXAPAL® and POWERBAKE® 4080. This synergy is observed when MAXAPAL® is added on both the sponge and dough sides. These results indicate that stronger synergy occurs when MAXAPAL® is added to the sponge side.

Example 11
A phospholipase A2 enzyme acting on N-acylphosphatidylethanolamine at the sn2 position is used in combination with an enzyme acting on polar lipids at the sn1 position to test the softness observed in corn bread (spong & dough) Experiment The effect of the addition of a phospholipase A2 enzyme (MAXAPAL®) acting on N-acylphosphatidylethanolamine at the sn2 position in combination with an enzyme acting on polar lipids at the sn1 position (POWERBAKE® 4090) The investigation was conducted in detail in corn bread (sponge and dough procedure) using MAXAPAL® added in the sponge step and POWERBAKE® 4090 added in the dough step.

  POWERBAKE (registered trademark) 4090 is an enzyme that acts on polar lipids at the sn1 position. In particular, POWERBAKE® 4090 is a fungal lipolytic enzyme having PLA1 activity to polar lipids and having SEQ ID NO: 1 disclosed herein. POWERBAKE® 4090 with an enzyme activity of 15,500 TIPU was used.

Experimental design:

  The use of SUREBAKE® 800 (HOX) and ascorbic acid remained constant at 50 and 60 ppm for all experiments, respectively.

  Two loaves of bread from each test variable were tested, and the softness (or hardness) of the sliced bread was determined from texture profile analysis (TPA) using a Texture Analyzer TAXT Plus from Stable Microsystems. A 35 mm metal probe was used on days 1 and 3.

Softness test results:
Separate testing of MAXAPAL (R) and POWERBAKE (R) 4090 respectively requires stronger power compared to controls (MAXAPAL (R) or POWERBAKE (R) 4090 not added) It showed that it was harder bread. The combination of MAXAPAL (R) and POWERBAKE (R) 4090 gave the best softness. The same combination also showed the highest synergy with volume (data not shown herein).

  The results of the bread softness test shown in FIG. 2 confirm that positive synergy was obtained by adding the combination of MAXAPAL® and POWERBAKE® 4090. This synergy was observed in the production of corn bread.

Example 12
The softness observed in 100% whole wheat bread (sponge & dough) using a phospholipase A2 enzyme acting on N-acylphosphatidylethanolamine at the sn2 position in combination with an enzyme acting on polar lipids at the sn1 position The baking experiment to test the length of the phospholipase A2 enzyme acting on N-acylphosphatidylethanolamine at the sn2 position (MAXAPAL®) in combination with the enzyme acting on polar lipids at the sn1 position (POWERBAKE® 4090) The effect of the addition was investigated in detail in the whole wheat (sponge and dough) bread procedure using MAXAPAL® added in the sponge stage and POWERBAKE® 4090 added in the dough stage.

Experimental design:

  The use of SYREBAKE® 800 (HOX) and ascorbic acid was kept constant at 100 and 100 ppm respectively for all experiments.

  Two loaves of bread from each test variable were tested, and the softness (or hardness) of the sliced bread was determined from texture profile analysis (TPA) using a Texture Analyzer TAXT Plus from Stable Microsystems. A 35 mm metal probe was used on days 1 and 3.

Softness test results:
PLA2 alone showed increased softness on both day 1 and day 3 as compared to the control (MAXAPAL® or POWERBAKE® 4090 not added).

  POWERBAKE® 4090 exhibited the same level of softness as control MAXAPAL® or POWERBAKE® 4090.

  The combination of MAXAPAL® and POWERBAKE® 4090 showed increased softness.

  The result of bread softness test in 100% whole wheat bread shown in FIG. 3 shows that MAXAPAL (R) alone is a control (MAXAPAL (R) or POWERBAKE (R) 4090 is not added) It has been shown to increase the softness compared to. However, the combination of MAXAPAL (R) and POWERBAKE (R) 4090 further increases the softness compared to MAXAPAL (R) alone, or POWERBAKE (R) 4090, or a control.

Example 13
Characteristics and baking performance of sn2-specific, NAPE-active PLA2 The purpose of these experiments is to investigate another phospholipase with sn2-specificity and NAPE activity (CRC 08 335) when combined with the sn1-specific enzyme POWERBAKE® 4080 It was to verify the synergistic performance in the baking application for

Method:
Gas chromatography (GLC)
Free fatty acids were analyzed by GLC as trimethylsilyl derivatives (TMS).

Sample Preparation The evaporated sample is dissolved in 1.5 mL of heptane: pyridine (2: 1). Transfer 500 μL of the sample solution to a crimp vial, add 100 μL of MSTFA (N-methyl-N-trimethylsilyl-trifluoroaceamide), and react at 60 ° C. for 15 minutes.

Cloning of CRC08335 The synthetic gene (CRC08335) encoding fungal phospholipase A2 type 2 is a gene which is codon optimized for expression in Trichoderma reesei, Generay (http://www.generay.com. It was ordered from cn / english /). The protein sequence of CRC08335 (SEQ ID NO: 4) (FIG. 5) was identified from an in-house Myceliophthora thermophile strain and is characterized by a thermotromeliasis comprising the closest homolog (NCBI Accession No. XP — 0036649499. 1) in the NCBI database. It shares 95% identity with Thermothelomyces thermophila secretory phospholipase A2 from ATCC 42464). CRC 08 335 has an N-terminal signal peptide sequence as predicted by Signal P 4.0 (Signal P 4.0: Identify the signal peptide from the transmembrane region: Thomas Nordahl Petersen, Soeren Brunak, Gunnar von Heijne & Henrik Nielsen. Nature Methods, 8: 785-786, 2011), which suggests that CRC08335 is an extracellular enzyme.

  The synthetic gene of CRC 08335 (SEQ ID NO: 5) (FIG. 6) carrying the N-terminal natural signal peptide is described in pGXT (International PCT application WO 2015/017256 pamphlet, incorporated herein by reference) (Identical to the pTTTpyr2 vector). In the pTTTpyr2 vector, the Aspergillus nidulans pyrG gene was transformed into H. coli. It is replaced with the H. jecorina pyr2 gene. The pTTT-pyr2 expression vector contained the Trichoderma reesei cbhI-derived promoter (cbhI) region and the cbhI terminator region, which can strongly induce the expression of the gene of interest. A. A. nidulans amdS and pyr2 selective markers confer growth of transformants on acetamide as sole nitrogen source, and the Trichoderma reesei telomeric region is non-nonspecific in fungal cells. Allows for chromosomal plasmid maintenance. After cloning of CRC08335, the resulting plasmid was designated pZKY512-1. The plasmid of pZKY512-1 is provided in FIG.

  The protein sequence of CRC08335 identified from an in-house Myceliophthora thermophile strain was defined as SEQ ID NO: 4. The polypeptide sequence of the predicted signal peptide is MKFLSTALCLASSVLA (SEQ ID NO: 6).

Transformation of CRC08335 The plasmid pZKY512-1 was transformed into a suitable Trichoderma reesei strain using protoplast transformation (Te'o et al. (2002) J. Microbiol. Methods 51: 393-99). Transformed (method described in published PCT patent application WO 05/001036). Transformants were selected on solid medium containing acetamide as sole nitrogen source. After 5 days of growth on acetamide plates, transformants were harvested and fermented in a 250 mL shake flask in synthetic medium containing a mixture of glucose and sophorose.

result:
SN1 / SN2 Specificity:
The enzyme specificity for CRC08335 was determined according to "Assay for determining phospholipase activity and sn1 and sn2 position specificity for PC (phosphatidyl choline)". This assay uses a PC substrate with a tailored FFA (free fatty acid) composition that analyzes released FFA by GLC analysis. The results are outlined in Table 16.

  The data in Table 16 show that MAXAPAL® and CRC 08335 are sn2-specific as indicated by the high "relative PLA2 activity (%)" compared to "relative PLA1 activity (%)" It shows clearly. Furthermore, the data clearly reflects that POWERBake® 4080 is sn1 specific.

NAPE activity:
The NAPE activity of CRC08335 was assessed by lipid profile analysis of the dough from baking trials performed with and without the enzyme. The baking application was carried out according to the procedure for hard crust roll (Example 1).

  NAPE activity was verified by HPLC analysis of dough lipids. The dough lipids were completely proofed according to the procedure for extraction of lipids from dough and extracted from freeze dried dough.

  The isolated lipids were analyzed by HPLC using a HILIC DIOL column, 1.7 μm, 50 × 2.1 mm (Fortis Technologies Ltd, UK). The solvents used are as follows: gradient: 100% solvent A to 100% solvent B for 20 to 20 minutes, 100% solvent B for 20 to 30 minutes, 100% solvent for 30 to 40 minutes Solvent A: 96% acetone, 4% methanol, 1 mM ammonium formate and solvent B: 60% acetone, 34% methanol, 6% MiliQ water, 1 mM ammonium formate. NAPE and NALPE were quantified using a charged aerosol detector and 1-palmitoyl-sn-glycero-3-phosphoethanolamine-N-linoeoil (Linoeoil) (Avanti Polar Lipids, Alabama, USA) as an internal standard. . The results from HPLC analysis are shown in Table 17.

  The results set forth in Table 17 clearly show that CRC 08335 has NAPE activity, as shown by the hydrolysis of NAPE and the formation of the more emulsifying component NALPE. POWERBake® 4080 also exhibits NAPE activity, as demonstrated by the hydrolysis of NAPE and the formation of the more emulsifying component NALPE. In addition to the generation of NALPE, POWERBake® 4080 also shows the generation of NAGPE (data not shown).

Applicable performance:
The synergistic application performance of the sn2-specific NAPE active enzyme CRC08335 in combination with POWERBake® 4080 was evaluated by a baking trial performed with and without the enzyme. The baking application was carried out according to the procedure for hard crust roll (Example 1).

  The synergistic application performance of CRC08335 and POWERBake® 4080 was demonstrated by the increased relative specific volume as outlined in Table 18.

  The results described in Table 18 indicate that, when used as a sole component, the sn2-specific NAPE active enzyme, CRC08335, has a limiting effect on the specific volume. The combination (at each dose) of CRC08335 and POWERBake® 4080 and the formation of the emulsifying component from both phospholipid and galactolipid hydrolysis show a clear synergy on the specific volume.

Conclusion Based on the baking experiments and analysis of dough lipids, surprisingly, POWERBAKE® 4080, or the combination of POWERBAKE 4090 and MAXAPAL® PLA2 phospholipase, or LYSOMAX® Oil Has been found to provide a positive synergy in firing. This is confirmed by the improvement of bread volume and the improvement of dough and bread characteristics including softness.

  Positive synergy on bread volume was also observed when MAXAPAL® was combined with other PLA1 enzymes such as, for example, LIPOPAN FTM and PANAMORE®.

  Baking tests with other PLA2 enzymes LIPOMODTM 699L and CRC08335 also showed positive synergy when combined with POWERBAKE® 4080.

  This synergy was confirmed in different baking experiments using different types of flour.

  POWERBAKE® 4080 and POWERBAKE® 4090 are both glycolipases with sn1 activity on galactolipids and phospholipids in dough. The PLA2 phospholipase MAXAPAL® hydrolyzes NAPE (and other phospholipids) at the sn2 position during the production of sn1-NALPE, but does not hydrolyze NALPE to a significant extent. NAPEs have different fatty acid compositions at the sn1 and sn2 positions, and will typically have more saturated fatty acids (C16: 0 and C18: 0) at the sn1 position. HPLC / MS analysis showed that MAXAPAL contributes to a strong increase of C16: 0_NALPE in dough.

  While not wishing to be bound by theory, C16: 0_NALPE is expected to show a stronger improvement in dough stability than C18: 2 NALP, but it depends on the fatty acid composition of NALPE in aqueous system To form various mesophases.

  However, MAXAPAL® by itself did not contribute to a large effect on the bread volume, but when combined with either POWERBAKE® 4080 or POWERBAKE® 4090, a strong synergy was obtained It is formed. This can be explained by the reaction products C16: 0_NALPE, MGMG and DGMG produced by the combination of the two enzymes.

  In some systems, MAXAPAL (R) and POWERBAKE (R) 4080 (or MAXAPAL (R) and POWERBAKE (R) 4090) compete for NAPE substrate when used in combination there is a possibility. This can be mitigated in certain baking procedures where the dough is mixed in two stages, for example in the so-called sponge and dough procedure. In this type of baking, add MAXAPAL® (or other PLA2 with activity to NAPE) on the sponge side for optimal generation of C16: 0_NALPE, then dow side for DGMG and MGMG generation It is possible to add POWERBAKE® 4080 or POWERBAKE® 4090 at. Yet another advantage of the addition of MAXAPAL® on the sponge side was the ready availability of reaction products (eg, NALPE) that contribute to the improvement of the properties of the dough during dough raking.

Claims (22)

  A food enzyme composition comprising: a phospholipase A2 enzyme that acts on N-acylphosphatidylethanolamine at the sn2 position; and an enzyme that acts on a polar lipid at the sn1 position.   A method of producing a dough comprising the steps of mixing a dough component, a phospholipase A2 enzyme acting on N-acylphosphatidylethanolamine at the sn2 position, and an enzyme acting on a polar lipid at the sn1 position.   Use of a phospholipase A2 enzyme acting on N-acylphosphatidylethanolamine at the sn2 position and an enzyme acting on a polar lipid at the sn1 position in the preparation of a dough or baked product, the specific volume of the baked product; properties of the dough ( Formation of dough; to improve the extensibility of the dough etc .; to improve the crispiness of the crust of the baked product; improve the crumb structure (to improve the crumb diameter of the baked product; or To improve homogeneity etc.); to improve softness (eg to improve softness of baked products); to improve oven springs of baked products; N-acyl lyso in said dough and / or baked products Increase phosphatidyl ethanolamine (preferably 14 to 20 carbon atoms Increasing the N-acyl lysophosphatidyl ethanolamine comprising fatty acid moieties, preferably increasing the N-acyl lysophosphatidyl ethanolamine comprising saturated fatty acid moieties containing 14 to 20 carbon atoms; And / or to increase the lysophospholipid in the baked product; to increase the digalactosyl monoglyceride and / or monogalactosyl monoglyceride in the dough and / or baked product; lysophospholipid in the dough and / or baked product, and / or Or use for increasing N-acyl lysophosphatidylethanolamine together with increasing digalactosyl monoglycerides and / or monogalactosyl monoglycerides.   The method according to claim 2 or the use according to claim 3, wherein the phospholipase A2 enzyme and the enzyme acting on polar lipids at the sn1 position are mixed or used either simultaneously or sequentially.   The phospholipase A2 enzyme converts N-acylphosphatidyl ethanolamine into N-acyl lysophosphatidyl ethanolamine under dough conditions, and the fatty acid portion of the generated N-acyl lysophosphatidyl ethanolamine is 14 to Containing 20 carbon atoms (preferably, said fatty acid part of said generated N-acyl lysophosphatidylethanolamine is saturated and contains 14 to 20 carbon atoms; more preferably, The composition according to claim 1, or the method according to claims 2 or 4, or the use according to claims 3 or 4, wherein said fatty acid is saturated and contains 16 carbon atoms .   The composition according to claim 1 or 5, or any one of claims 2, 4 or 5, wherein the enzyme acting on polar lipids at the sn1 position is an enzyme having phospholipase activity, galactolipase activity or a combination thereof. The method according to any one of the claims 1 to 7 or the use according to any one of claims 3 to 5.   The composition according to any one of claims 1, 5 or 6, or any one of claims 2 or 4 wherein the enzyme acting on polar lipids at the sn1 position acts on galactolipids. The method according to claim 1 or the use according to any one of claims 3 to 6.   The composition according to any one of claims 1 or 5 or 7, wherein said phospholipase A2 enzyme is unable or not substantially to act on N-acyl lysophosphatidylethanolamine A method according to any one of claims 2 or 4-7, or a use according to any one of claims 3-7.   A composition according to any one of claims 1 or 5 to 8, wherein the composition further comprises lecithin, preferably a lecithin based on soy.   A method according to any one of claims 2 or 4 to 8, or any one of claims 3 to 10, further comprising the step of adding lecithin, preferably adding a soy based lecithin. Use described in.   The composition according to claim 9, or the use according to claim 10, or the method according to claim 10, wherein the lecithin is an enzymatically modified lecithin.   The lecithin is enzymatically modified by an enzyme with phospholipase A2 activity (preferably, the lecithin is enzymatically modified by a phospholipase A2 which acts on the N-acylphosphatidylethanolamine at the sn2 position) A composition according to claim 9 or 11, a method according to claim 10 or 11, or a use according to claim 10 or 11.   The method further comprising the step of cooking, eg baking or steaming, boiling or frying the dough to produce a product, eg a baked or steamed product, or a boiled or fried product. The method according to any one of claims 8 to 10 or the use according to any one of claims 3 to 8 or 10 to 12.   The method according to any one of claims 2, 4 to 8 or 10 to 13, wherein the dough is a dough selected from the group consisting of bread dough, pasta dough, noodle dough, cake dough, pastry dough or batter, or 14. Use according to any one of claims 3-8 or 10-13.   The phospholipase A2 enzyme, and the enzyme acting on polar lipids, are under the same conditions except for the addition of the claimed enzyme of a baked product, or a steamed product, or a braised product, or a fried product manufactured from the dough The dough component is mixed with the dough component in an effective amount to produce an increase in specific volume which is at least 10% compared to a baked or steamed product, or a steamed product, or a braised product, or a fried product, according to The method according to any one of 4-8 or 10-13, or the use according to any one of claims 3-8 or 10-13.   The phospholipase A2 enzyme is present at a concentration of 50 to 7500 ePLU / kg (powder), preferably 150 to 2000 ePLU / kg (powder), more preferably 500 to 1000 ePLU / kg (powder). A composition according to any one of the claims, or a method according to any one of the claims 2, 4-8 or 10-15, or any one of the claims 3-8 or 10-15 Use of.   The composition according to any one of claims 1, 5-9 or 16, or the enzyme according to any one of claims 1, 5-9 or 16, wherein the enzyme acting on polar lipids at the sn1 position is present at a concentration of 100 to 2000 ePLU / kg (powder). , 4-8 or the method according to 10-16, or the use according to any one of claims 3-8 or 10-16.   18. A composition according to any one of claims 1, 5-9 or 17 or any one of claims 2, 4-8 or 10-17, wherein an additional enzyme is added to said composition or dough. 18. A method according to paragraph or a use according to any one of claims 3-8 or 10-17.   Said further enzymes include lipases, starch degrading enzymes (eg amylase or amyloglucosidase), hemicellulases (eg xylanases), cellulases, oxidoreductases (eg glucose oxidase or hexose oxidases), lipid acyltransferases, debranching enzymes (eg The method according to claim 18, or the composition according to claim 18, or the use according to claim 18, which is, for example, one or more selected from the group consisting of pullulanase), lactase and protease.   20. The method according to any one of claims 2, 4-8 or 10-19, wherein the amount of C16: 0 NALPE in the dough is increased by at least 1.5 times compared to the dough without added enzyme. Or a use according to any one of claims 3-8 or 10-19.   The sponge and dough method wherein said phospholipase A2 enzyme capable of acting on NAPE is added to sponge and said enzyme acting on polar lipid at said sn1 position is added to said dough. The method according to any one of claims 8 to 10 or the use according to any one of claims 3 to 8 or 10 to 20.   A kit comprising a phospholipase A2 enzyme capable of acting on N-acylphosphatidylethanolamine at the sn2 position, an enzyme acting on a polar lipid at the sn1 position, and a set of instructions for use.

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